ff 2,5
January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
DINOSEB
I
(X
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460.
&
HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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TABLE OF CONTENTS
Page
LIST OF FIGURES .. . ^ v
LIST OF TABLES . . v
FOREWORD vi
AUTHORS, CONTRIBUTORS, AND REVIEWERS vii
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES ....... II-l
A. Genera] Properties -.*..... II-l
B. Manufacture and Use "... II-I
C. Environmental Effect and Stability. II-l
D. Summary . II-3
III. TOXICOKINETICS III-l
A. Absorption III-l
B. Tissue Distribution III-2
C. Metabolism III-5
D. Excretion III-8
E. Bioaccumulation and Retention 111-10
F. Summary 111-10
IV. HUMAN EXPOSURE IV-1
V. HEALTH EFFECTS IN ANIMALS V-l
A. Short-term Exposure V-l
1. Lethality V-l
2. Other Acute Effects .- V-5
B. Long-term Exposure V-7
1. Subchronic Effects V-7
2. Chronic Effects V-9
C. Developmental/Reproductive Effects V-10
1. Developmental Effects V-10
2. Reproductive Effects V-23
D. Mutagenicity V-26
1. Gene Mutation Assays (Category 1) V-26
2. Other Genotoxic Effects (Category 3) V-28
E. Carcinogenicity V-29
F. Summary V-31
iii
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TABLE OF CONTENTS (continued)
Pace
VI. HEALTH EFFECTS IN HUMANS ........ VI-1
A. Clinical Case Studies ' VI-1
B. Epidemiological Studies VI-2
C. High-Risk Populations VI-3
D. Summary VI-3
VII. MECHANISMS OF TOXICITY VII-1.
A. Uncoupling of Oxidative Phosphorylation VII-1
B. Methemoglobin Formation VII-2
C. Interactions VII-2
D. Summary VII-2
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
A. Procedures for Quantification of Toxicological Effects. . VIII-1
1. Noncarcinogenic Effects VIII-1
2. Carcinogenic Effects . VIII-4
B. Quantification of Noncarcinogenic Effects for Oinoseb . . VIII-6
1. One-day Health Advisory VIII-6
2. Ten-day Health Advisory VIII-6
3. Longer-term Health Advisory VIII-7
4. Reference Dose and Drinking Water Equivalent Level . VIII-8
C. Quantification of Carcinogenic Effects for Dinoseb. . . . VIII-9
D. Existing Guidelines VIII-10
E. Summary VIII-11
IX. REFERENCES IX-1
iv
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LIST OF FIGURES
Figure No. Page
III-l Metabolic Pathways of Dinqseb in Mammals Based on
Reported Metabolites III-8
LIST OF TABLES
Table No.
II-l Properties of Dinoseb (2-sec-butyl-4,6-dinitrophenol) . II-2
III-l Percutaneous Absorption of Dinoseb in the Rhesus
Monkey III-3
II1-2 Mean Tissue 14C .Levels of Dinoseb and Metabolites 3
Hours After Oral or Intraperitoneal Administration to
Pregnant Mice '. III-4
II1-3 Excretion of 14C-Dinoseb by Female Mice Following
Oral or Intraperitoneal Administration. ... III-9
V-l Acute Oral Toxicity of Dinoseb V-2
V-2 Acute Dermal Toxicity of Dinoseb V-4
V-3 Effect of Dinoseb on Pregnancy Performance in Rats. . . V-13
V-4 Summary of Body Weight Changes in Pregnant Rabbits
Percutaneously Treated With Dinoseb During Days 7
to 19 of Gestation V-19
V-5 Summary of Embryo/Fetal Toxicity in Pregnant Rabbits
Percutaneously Treated With Dinoseb During Days 7 to
19 of Gestation V-21
V-6 Incidences (%) of Litter and Fetal Malformations Found
in Rabbits Percutaneously Treated With Dinoseb During
Days 7 to 19 of Gestation V-22
V-7 Epididymal Sperm Counts in Rats Fed Dinoseb for 71 to
77 Days V-25
V-8 Incidence of Hepatocellular Adenoma and Carcinoma in
Mice Receiving Dinoseb in the Diet for 100 Weeks. . . . V-30
VIII-1 Summary of Quantification of Toxicological Effects
for Dinoseb VIII-12
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended In 1986,
requires the Administrator of the Environmental Protection Agency to publish
Maximum Contaminant Level Goals (MCLGs) and promulgate National Primary Drinking
Water Regulations for each contaminant, which, In the judgment of the
Administrator, may have an adverse effect on public health and which is known or
anticipated to occur in public water systems. The HCLG is nonenforceable and is
set at a level at which no known or anticipated adverse health effects in humans
occur and which allows for an adequate margin of safety. Factors considered in
setting the MCLG include health effects data and sources of exposure other than
drinking water.
This document provides the health effects basis to be considered in
establishing the MCLG. To achieve this objective, data on pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemiology,
and mechanisms of toxicity were evaluated. Specific emphasis 'is placed on
literature data providing dose-response information. Thus, while the literature
search and evaluation performed in support of this document was comprehensive,
only the reports considered most pertinent in the derivation of the MCLG are
cited in the document. The comprehensive literature data base in support of this
document includes information published up to April 1987; however, more recent
data have been added during the review process and in response to public
comments.
When adequate health effects data exist, Health Advisory values for less-
than-lifetime exposures (One-day, Ten-day, and Longer-term, approximately 10% of
an individual's lifetime) are included in this document. These values are not
used in setting the MCLG, but serve as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
James R. Elder
Director
Office of Ground Water and Drinking Water
Tudor T. Davies
Di rector
Office of Science of Technology
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I. SUMMARY
Oinoseb, 2-sec-butyl-4,6-dinitrophenol (DNBP), is poorly soluble in water
(0.52 g/L) but is readily soluble in alcohol and other organic solvents. DNBP
is often used in salt form as both a pre- and postemergence herbicide for a
wide variety of crops.
About 25 to 37% of an oral dose and approximately 40% of an intraperi-
toneal dose of dinoseb is excreted in the feces (the remainder being
eliminated in the urine or retained in tissues), suggesting that fecal
excretion results primarily from secretion of absorbed dinoseb into the
intestines. On the basis of this information, absorption may be estimated to
be essentially complete. A maximum of approximately 7% of the administered
dose was found in the urine of monkeys following percutaneous administration
of dinoseb. When administered orally to pregnant mice, absorbed dinoseb
and/or dinoseb residues are distributed to all tissues. Residue levels in the
brains and embryos of pregnant mice never exceeded 2.5% of the plasma level.
Dinoseb is extensively metabolized by several pathways in animals: (1) one or
both of the nitro groups can be reduced to the amine, which may then be
acetylated; (2) the terminal methyl groups of the side chain can be oxidized
to carboxyl groups; and (3) the compound or its metabolites may be conjugated,
primarily as glucuronides. Although a number of metabolites have been
identified, a greater number have been detected but not yet characterized.
Dinoseb and its metabolites are excreted in both urine and feces, with low
amounts in the bile. Excretion was monophasic following a single oral dose,
with a half-time of about 35 hours. A t,/2 of about 8 hours was observed
following an intraperitoneal dose.
.Acute oral LDSO values for the rat, rabbit, mouse, and guinea pig range
from 14 to 114 mg/kg. The intraperitoneal LDSO value has been reported as
20.2 mg/kg for female mice and 10 mg/kg for male mice. Dinoseb is well
absorbed through intact skin, with dermal LD50 values in the rat ranging from
67 to 134 mg/kg.
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When administered intraperitoneally, dinoseb doses of 12 to 16 mg/kg/day
for 5 days intensified inhibitory and excitatory activities in the brains of
rats, while doses of 2 to 8 mg/kg/day for 5 days were without effect. In
ducklings, dinoseb and a number of other dinitrophenols have the ability to
produce cataracts following dietary exposure.
Increased mortality was noted in rats fed diets containing dinoseb at 300
ppm and above for 60 days. Decreased body weight gains were noted at dietary
levels of 50 to 200 ppm. Diffuse tubular atrophy of the testes was noted in
males receiving 200 ppm. In a 6-month feeding study, the body weights of rats
receiving 5.4 mg/kg/day were slightly lower than those of controls at the end
of the treatment period. No other toxic effects were noted in these animals,
except for a slight but statistically significant increase in liver weight.
Dietary levels of 13.5 mg/kg/day caused increased mortality, whereas 2.5
mg/kg/day was a No-Observed-Adverse-Effect Level (NOAEL).
No adverse effects were noted in beagles administered diets containing
0.01 or 0.005% dinoseb for 91 days. However, in females fed dietary levels of
0.02 and 0.03%, growth retardation, increased average liver weights, mural
endocarditis, and microscopic heart changes were noted. The NOAEL was
established at 0.01% (100 ppm), equivalent to 4 mg/kg/day.
A compound-related decrease in mean thyroid weights was noted in rats
receiving dietary levels of 1, 3, and 10 mg/kg/day for 2 years. No other
compound-related effects were noted, but histopathologic examination of
tissues was conducted in only a limited number of animals. A Lowest-Observed-
Adverse-Effect Level (LOAEL) of 1 mg/kg/day was established from this study.
Mice orally administered dinoseb for 100 weeks at dietary levels of 1, 3,
and 10 mg/kg/day showed cystic endometrial hyperplasia and atrophy, hyposperm-
atogenesis, and testicular degeneration, but oncogenic effects were equivocal.
Statistically significant increases in liver adenomas and adenomas plus
carcinomas were observed in female mice only. Lenticular opacities were
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observed at the 3- and 10-mg/kg dose levels, but animals receiving the low
dose were not examined. A systemic NOAEL is less than 1 mg/kg/day.
Dinoseb has been found to be teratogenic in several species including
rabbits, rats, and mice following oral, dermal, intraperitoneal, and
subcutaneous administration to pregnant animals. Oral administration of
dinoseb in mice on day 8 of gestation, at doses of 26 and 33 mg/kg, produced
supernumerary ribs. The same anomaly was seen in rats administered 10 mg/kg
dinoseb on days 6 to 15 of gestation. Skeletal anomalies, as well as external
and visceral malformations, were also observed in rabbits orally administered
dinoseb on days 6 to 18 of gestation at the same dose of 10 mg/kg.
Oral administration of dinoseb on days 10 to 12 of gestation produced
skeletal anomalies at 32 mg/kg/day. Some anomalies occurred at 20 mg/kg, but
these were considered marginal. Treatment of pregnant mice with 17.7 mg/kg
dinoseb administered intraperitoneally on days 10 to 12 of gestation resulted
in fused or missing ribs, fused or missing sternebrae, fused or unossified or
absent vertebrae, and absent or unossified long bones. Subcutaneous doses
produced comparable effects, but these were observed at somewhat higher dose
levels.
The developmental anomalies appeared at lower dosages of dinoseb after
dermal exposure than after oral administration. In a developmental toxicity
study in rabbits, a developmental NOAEL of 1 mg/kg was identified based on
increases in gross external, soft tissue, and skeletal malformations in the
fetuses of dams given 3 or 9 mg/kg/day percutaneous!y on days 7 through 19 of
gestation. These malformations included hydrocephaly, microphthalmia,
anophthalmia, craniosynostosis, and small eye sockets. The maternal NOAEL was
also 1 mg/kg/day based on increased mortality, slight decreases in body
weights during the dosing period, and increased incidences of gross lesions
upon necropsy (hemorrhaging in brain, trachea, thymus, lungs, and subdermis of
the thorax and abdomen) of rabbits receiving dosages of 3 mg/kg/day or higher.
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The teratogenic effects noted in mice following intraperitoneal adminis-
tration of dinoseb are diminished by its metabolism. Compounds that stimulate
drug metabolism (such as phenobarbital) have been shown to decrease dinoseb
toxicity. Conversely, inhibitors of drug metabolism (such as SKF-525A)
potentiate dinoseb-induced teratogenicity.
Other studies suggest that the rat may lie more susceptible than the mouse
to the effects of dinoseb. Pregnant Sprague-Dawley rats fed 9.23 mg/kg/day of
dinoseb in the diet on days 6 to 15 of gestation had poor weight gain, ataxia,
and lethargy. At all dose levels above 8.60 mg/kg/day, there was a
significant reduction in fetal survival per litter at birth.
Results of a study of the postnatal morphology and functional capacity of
kidneys in neonates of Sprague-Dawley rats treated intraperitoneally with
dinoseb show that approximately 40% of the fetuses of mothers treated with
dinoseb at 8 to 9 mg/kg/day had dilated renal pelves and/or ureters.
Histological examination revealed relatively complete recovery at 6 weeks of
age. In contrast, livers of fetuses from this same group showed highly
vacuolated cells that were still present in offspring 6 weeks later, along
with necrotic cells and pyknotic or karyorrhectic nuclei in other cells, thus
demonstrating little evidence of recovery.
In mice orally administered 15 and 100 mg/kg during days 8 to 12 of
gestation, no effects were seen on postnatal parameters at day 22 or 30.
In a three-generation rat study in which dinoseb was administered at 1,
3, and 10 mg/kg/day, no effect on survival, fertility, or fecundity was seen.
At 10 mg/kg/day, the number of pups born and the pup weights at weaning were
lower and were attributed to maternal toxicity.
In an 11-week study with rats, dinoseb at dietary levels of 15.6 or
22.2 mg/kg/day produced marked oligospermia and extensive loss of spermato-
genic cells. Little recovery occurred during 16 weeks following cessation of
exposure. At 9.1 mg/kg/day, decreased epididymal sperm counts, atypical
1-4
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epidldymal spermatozoa, and minimal testicular changes were present that
appeared to be reversible. No effects were seen In rats fed 3.8 mg/kg/day for
11 weeks. In a study on testicular toxicity of dinoseb in mice, daily
intraperitoneal injections of 20 mg/kg/day for 5 consecutive days produced no
testicular effects.
A number of assays were conducted to determine the mutagenic potential of
dinoseb. Negative responses were elicited in the Ames assay with
Salmonella tvphimurium and Escherichia coli. sex-linked recessive lethal assay
in Drosoohila melanoaaster. mitotic recombination assay in Saccharomvces
cerevisiae. and unscheduled DNA synthesis assay in human fibroblasts.
However, positive responses were elicited in DNA repair synthesis-assays using
repair-deficient and repair-proficient strains of £. coli. Bacillus subtil is.
and S. tvphimurium. Dinoseb also induced small increases in mutation
frequencies in a mouse lymphoma cell line.
No increases in the incidence of tumors were found in rats fed diets
containing 1, 3, or 10 mg/kg/day of dinoseb for 104 weeks. However, limited
histopathology was performed for this study; thus, the oncogenic potential was
difficult to assess. Mice orally administered dinoseb for 100 weeks at
dietary levels of 1, 3, and 10 mg/kg/day showed equivocal oncogenic effects,
although statistically significant increases in the incidence of liver
adenomas and combined adenomas and carcinomas were observed in female mice
only, A systemic NOEL of less than 1 mg/kg/day was calculated based on
increased incidences of cystic endometrial hyperplasia and atrophy/hyposperma-
togenesis/degeneration in the testes of dosed animals.
Only one case study of dinoseb poisoning in humans was identified in the
available literature. Signs of toxicity appeared shortly after the individual
had applied the compound to a field. The individual wore a gauze face mask
but not gloves as he repaired plugged spray-jets in the field. Thus, both
skin and inhalation exposure may have been extensive. Elevated body
temperature, liver damage, and subsequent lung involvement were the major
effects. The liver damage appeared to be particularly long lasting.
1-5
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No relevant epidemic!ogical studies were located. Estimates of dermal
and inhalation exposure in applicators were reported to be 33.7 and
0.12 mg/70-kg man/hour. Oinoseb has been detected in the blood of workers
employed in the manufacture of the herbicide at levels ranging from 0.0 to
0.2 ng/mi blood.
The toxicity of dinoseb was suggested to be due to the uncoupling of
oxidative phosphorylation. Experimental studies demonstrated that dinoseb
inhibition of brain oxidative phosphorylation correlated with signs and
symptoms of toxicity. In mice showing severe signs of poisoning, oxidative
phosphorylation of brain mitochondria was completely inhibited.
There were no suitable studies available for calculation of the One-day
Health Advisory. Thus, the Ten-day HA value was used as a conservative
estimate for the One-day HA. A developmental NOAEL of 3.0 mg/kg/day, based on
absence of teratogenic effects in fetuses of pregnant rabbits exposed by oral
administration on days 6 to 15 of gestation, was used to calculate the Ten-day
HA value of 300 *g/L for a 10-kg child. A Lowest-Observed-Adverse-Effect
Level (LOAEL) of 1.0 mg/kg/day, based on a decrease in pup body weight at all
levels in a two-generation study, was employed to calculate the Longer-term HA
of 10 ng/l for a 10-kg child and 40 »g/L for a 70-kg adult. Both a 100-week
feeding study with mice and a 2-year feeding study with rats indicate NOAEL
levels below 1 mg/kg/day. Treatment-related cystic endometrial hyperplasia
and atrophy, hypospermatogenesis, and degeneration of the testes were noted in
dosed mice, whereas decreased thyroid weights were noted in rats. Therefore,
a LOAEL of 1 mg/kg/day was selected to calculate the Reference Dose (RfO) and
Drinking Water Equivalent Level (DUEL). The RfD is calculated to be
1 (tg/kg/day, and the DWEL is 40 #g/L based on these studies.
No calculation of excess cancer risk has been made, since only equivocal
long-term effects of dinoseb carcinogenicity have been reported. The only
standards or guidelines found were the EPA RfD Work Group approval of a 0.001-
mg/kg/day RfD for dinoseb (U.S. EPA, 1987a) and a published tolerance (U.S.
EPA, 1986b) for dinoseb of 0.1 ppm for a wide variety of agricultural
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commodities. EPA has Issued a notice of intent to cancel registration of
pesticide products containing dinoseb and dinoseb salts (U.S. EPA, 1986c), as
well as an emergency suspension of pesticide products containing dinoseb salts
(U.S. EPA, 1986d).
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II. PHYSICAL AND CHEMICAL PROPERTIES
A. GENERAL PROPERTIES
Dinoseb is 2-sec-butyl-4,6-dlnitrophenol (DNBP). The phenol form is
poorly soluble in water (0.52 g/L), but is readily soluble in most organic
solvents. The major physical and chemical properties are summarized in
Table II-l.
B. MANUFACTURE AND USE
Dinoseb can be synthesized by nitration of 0-sec-butyl phenol,-or by
sulfonation of phenol to block the p-position, followed by butylation and
removal of the sulfonic group (Spencer, 1982). U.S. production of dinoseb was
reported to be 6.2 million pounds/year in 1982 (CEH, 1985).
A variety of dinitrophenols are used extensively as herbicides and
pesticides. Dinoseb and its salts are used as selective weed killers in field
crops and pastures and along roadsides and rights-of-way. Dinoseb ammonium
salt is used as a postemergence, selective spray in flax, beans, peas, leeks,
potatoes, coffee, vineyards, and orchards. The alkanolamine salt is often
used as a preemergence and early postemergence spray (Call et al., 1983). It
has been reported that the triethanolamine salt, which is a common
formulation, may contain as much as 260 ppm of N-nitrosodiethanolamine (Zweig
et al., 1980).
C. ENVIRONMENTAL EFFECT AND STABILITY
Malkomes and Wohler (1983) reported on the effects of dinoseb on micro-
organisms in two types of soil. Dehydrogenase activity, adenosine triphos-
phate (ATP) content, and carbon dioxide evolution were used to measure the
effect of the herbicide on soil organisms. In laboratory studies, three
vessels were filled with about 1 kg of soil, and dinoseb (429 g dinoseb
acetate/L) was mixed into the soil at a level equivalent to application of
4 L/hectare. The soils were incubated in the laboratory at 10 or 20°C for
II-l
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Table II-l. . Properties of Dinoseb (2-sec-butyl-4,6-dinitrophenol)
Property
Value
Molecular formula
Structure
Molecular weight
Physical appearance
Melting point
Density
Vapor pressure
Solubility (g/100 ml solvent)
Water
Ethanol
Ethyl Ether
n-Heptane
Toluene
Xylene
C,oH12N2°s
240.2
Dark amber crystals
32°C
1.2647 at 45°C
(15rCJ 1 mmHg
(262°C) 100 mmHg
0.052
48
Miscible
27
Miscible
Miscible
SOURCE: Adapted from the Weed Science Society of America (1983).
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periods of up to 16 months. Moisture content was maintained at 40 or 60%,
respectively. A marked depression of dehydrogenase activity, ATP content, and
carbon dioxide production occurred within 1 to 4 weeks, and this inhibition
was still evident after several months. Field studies showed fairly com-
parable results with respect to dehydrogenase activity (which was still
depressed after 2 to 4 months), but other parameters behaved somewhat dif-
ferently. Carbon dioxide evolution in treated soils varied only slightly from
that in control soils, and ATP levels, which sometimes were higher in treated
than in control soils, were unpredictable. The authors pointed out the
difficulty of predicting the interactions of soil organisms and chemicals.
Hawkins and Saggers (1974) coated eight apples on a tree with 14C-labeled
dinoseb to study retention times under environmental conditions; each apple
was exposed to 30 ng of the herbicide. The fruit was harvested at various
times (0.5 hours to 28 days) after treatment, and the skin and flesh of the
apples were analyzed for radioactivity. Approximately 72% of the dinoseb was
lost from the apples by 28 days. The maximum amount of dinoseb absorbed
through the skin was 7%. Eight hours after application, essentially all of
the residual dinoseb was present on the apple skin in the unaltered state.
However, measurements at 8 days and 28 days indicated that most of the label
was present on the apple skin in the form of degradation products, indicating
transformation of this compound under environmental conditions.
D. SUMMARY
Dinoseb, 2-sec-butyl-4,6-dinitrophenol, is poorly soluble in water
(0.52 g/L) but readily soluble in organic solvents. Dinoseb and its salts are
used as pre- and postemergence herbicides for a wide variety of crop and
noncrop applications. Dinoseb has a relatively long retention time in the
environment, although evidence shows that it is degraded when exposed to
sunlight and other environmental conditions.
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III. TOXICOKINETICS
A; ABSORPTION
Bandal and Casida (1972) administered a. single oral-dose of 8 to
10 ftmol/kg (1.9 to 2.4 mg/kg) of 14C ring-labeled DNBP (99% pure) to male
albino rats (180 g) and mice (20 g). After 72 hours, cumulative fecal
excretion was 25% of the dose in the rat and about 37% of the dose in the
mouse. Elimination in the urine accounted for 64 and 37%, respectively, in
the rat and mouse. From these data, absorption may be estimated to be a
minimum of 75 and 63% in the rat and mouse, respectively.
Gibson and Rao (1973) administered an oral dose of 32 mg/kg of uniformly
ring-labeled 14C-dinoseb to female Swiss-Webster mice. The rate constant for
gastrointestinal absorption was estimated to be 7 .+ 4 hour"1 (corresponding to
a t1/2 of 5.9 minutes). In nine mice, cumulative fecal excretion after
64 hours was 30.4% of the dose, while excretion in bile was 1.4%, suggesting
that absorption of dinoseb was approximately 70%. However, fecal excretion
was 41% following an intraperitoneal dose (17.7 mg/kg), suggesting that fecal
excretion results primarily from biliary excretion of absorbed dinoseb into
the intestines. Since the amount in the feces following parenteral dosing
(41%) more than accounts for the amount in feces following oral dosing
(30.4%), gastrointestinal absorption of dinoseb appears to be essentially
complete.
Froslie and Karlog (1970) gave two cows 15 g dinoseb via intraruminal
intubation. Within 5 minutes after administration, DNBP could be detected in
the plasma at levels as high as 5 to 10 pg/mL. Ten days later, 1 to 2 itg/ml
of the parent compound persisted in the plasma.
The dermal absorption of 14C-dinoseb was determined following application
of a single percutaneous dose of 0.045, 0.2, or 3.6 mg/cm2 to a shaved area on
the abdomen of female rhesus monkeys (4 to 10 kg body weight, four per group)
(Bucks, 1987). An additional four monkeys received single intravenous
III-l
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Injections of 3.0 mg 14C-d1noseb. Each dose contained approximately 5 pCi of
radioactivity. After a 24-hour exposure period, the test site was washed to
remove the remaining test material, and the monkeys were observed for an
additional 13 days during which urine, feces, and blood were collected at
specific intervals. The dermal absorption values, determined by measurement
of the amount of total radiolabel excreted in the urine for 14 days, were
approximately 5.4, 7.2, and 4.9% of the total radioactivity administered at
the low-, mid-, and high-dose levels, respectively {Table III-l). This
suggests that maximum percent absorption occurred at 0.2 mg/cm2 and suggests
that there was no further increase in absorption of dinoseb above 0.2 mg/cm2.
Bough et al. (1965) reported that dinoseb (technical grade) is readily
absorbed through the skin. In four rabbits treated with a dermal application
of 50 mg/kg, blood levels rose from 0 mg/100 ml to 6 to 8 mg/100 mL within 2
to 6 hours (resulting in death), but no quantitative estimate of dermal
absorption was provided.
B. TISSUE DISTRIBUTION
Gibson and Rao (1973) administered uniformly labeled 14C-dinoseb to
pregnant Swiss-Webster mice. Animals received doses of 17.7 mg/kg intra-
peritoneally or 32 mg/kg by stomach tube on day 11 of gestation. Animals.were
sacrificed at various time intervals, and tissues (including embryos,
placenta, and uterus) were taken for analysis. Radioactivity was present in
all tissues examined, but data were presented for only a few tissues and blood
plasma. The total WC residues and unchanged dinoseb in the embryo and blood,
liver, and kidneys of pregnant mice 3 hours after oral or intraperitoneal
dosing are shown in Table III-2. Both metabolites and parent dinoseb were
found in embryos, but embryonic levels examined at various intervals (1 minute
to 48 hours) after dosing never exceeded 2.5% of the maternal plasma levels.
Brain radioactivity was of the same order of magnitude as that in the embryo,
indicating a blood-brain barrier for dinoseb. The volume distribution of
dinoseb also appeared to depend on the route of administration. After oral
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Table III-l. Percutaneous Absorption of Dinoseb In the Rhesus Monkey
Dose Absorption
Appl. Total Max. abs.
Dose Dose rate Percent
(rag/cm2) (mg) (% dose/hr) absorption
0.2 2 2.1 5.4
0.045 0.45 0.15 7.2
3.6 32 0.14 4.9
*
SOURCE: Adapted from Bucks (1987). -
III-3
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Table II1-2. Mean Tissue "C Levels* of Dinoseb and Metabolites
3 Hours After Oral or Intraperitoneal Administration
to Pregnant Mice
Tissue
Blood
Liver
Kidney
Embryo
Total 14C
Oral
(32 mg/kg)
31.3 ± 5.4
26.5 ± 4.1
21.8 ± 3.9
2.1 ± Q.Z.
ip
(17. 7 mg/kg)
45.0 ± 1.4s
32.9 ± 1.3
28.6 ± 4.4
5.1 ± 0.4°
Parent
Oral
(32 mg/kg)
29.7 ± 4.6
14.2 ± 0.6
13.8 ± 1.4
1.8 ± 0.1
dinoseb"
ip
(17.7 mg/kg)
46.6 + 3.9
5.6 ± 1.8
15.4 ± 1.4
2.9 ± 0°
'Values are means ± SEM for at least three animals. Results are expressed
as *ig dinoseb/g tissue.
"Measured as methyl ethyl ketone-extractable material.
°p =» o.OS (oral versus intraperitoneal).
SOURCE: Adapted from Gibson and Rao (1973).
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administration, di.noseb was distributed in total body water, but after
intraperitoneal administration, it was distributed only in extracellular
water.
Hall et al. (1978) administered technical grade dinoseb (80%) at 0, 50,
100, 150, 200, 300, 400, or 500 ppm in the diet to Sherman rats (35 to 38 days
old) for 60 days. Tissue analysis revealed dose-related tissue residue levels
with blood>feces>adipose>brain>liver. Other information was not presented.
C. METABOLISM
Gibson and Rao (1973) also investigated the metabolism of dinoseb in
pregnant Swiss-Webster mice. Pregnant mice were dosed orally (32 mg/kg) or
intraperitoneally (17.7 mg/kg) on day 11 of gestation with uniformly ring-
labeled dinoseb. After 3 hours, animals were sacrificed, and blood, liver,
kidney, and embryos were analyzed for the presence of total label and parent
dinoseb (methylethyl ketone extractable). The difference between the two
values was attributed to metabolites. The data (shown in Table III-2)
indicate that dinoseb was metabolized by pregnant mice, and a greater per-
centage of metabolites was present in tissues (including the embryo) after
intraperitoneal dosing than after oral dosing. Specific metabolites were not
identified.
Ernst and Bar (1964) studied the metabolites of dinoseb in the urine of
rats and rabbits. Rabbits received 15.8 or 20.0 mg/kg DNBP as a single oral
dose, and rats received 5.8, 8.9, or 11.5 mg/kg daily. Compounds were
identified by paper chromatography in three different solvent systems and by
infrared spectroscopy. A small amount of unchanged DNBP (1.9 to 2.7%) was
found in the urine of both rats and rabbits. In both the rabbits and rats,
2-(3-butyric acid)-4,6-dinitrophenol, in which the terminal methyl group of
the side chain was oxidized to a carboxyl group, was detected; concentrations
were higher in the rabbits (10 to 14% of the dose) than in the rats (about
6%). Only the rabbits excreted 2-sec-butyl-4-nitro-6-aminophenyl-0-glu-
curonide. Total identified metabolites accounted.for approximately 15 to 27%
III-5
-------
of the dose administered to the rabbits and approximately 6% of the dose
administered to the rats. An unidentified compound (substance IV) constituted
15 to 18.5% of the radioactivity in the urine of the rabbits and up to 8% in
that of the rats. Ernst (1968) reviewed the metabolic data available on
dinoseb and proposed a metabolic scheme for the compound.
Bandal and Casida (1972) studied the metabolism of ON6P in rats and mice.
This report also included data on the metabolism of 2-sec-buty1-4,6-dinitro-
phenyl isopropyl carbonate (dinobuton). Male albino mice (20 g) and rats (180
g), strains not specified, were dosed by stomach tube with 8 to 10 jtmol/kg
(1.9 to 2.4 mg/kg) of 14C-labeled DNBP dissolved in dimethyl sulfoxide or
methanol. Treated animals were held in individual metabolism cages for 72
hours, with food and water ad libitum. In both rats and mice, the major
portion of a dose of dinobuton was rapidly hydrolyzed to dinoseb, after which
both compounds were metabolized by the same pathway. Oxidation of either of
the two methyl groups in the sec-butyl moiety may occur, yielding
2-(2-butyric acid)-4,6-dinitrophenol or 2-(3-butyric acid)-4,6-dinitrophenol.
The latter was found in the conjugated form in the rat, but not in the mouse.
The ortho nitro group may be reduced to yield 2-sec-butyl-4-nitro-6-amino-
phenol, which may exist in both free and conjugated forms. Approximately 12
other metabolites were detected but not identified. An additional unknown
complex, consisting of at least five metabolites that remained uncleaved by
beta-glucurdm'dase hydrolysis in the rat, accounted for 70 and 52% of the
radiolabel recovered in the urine of the mouse and rat, respectively.
Froslie and Karlog (1970) studied the metabolism of ON6P in the cow. A
dose of 15 g was administered by tube to the rumen. This was nearly fatal,
but the animal slowly recovered over a 2-week period. Analysis of products in
the rumen indicated that within 30 minutes, DNBP was converted primarily to
6-amino-NBP. The 6-amino-NBP, in turn, was gradually converted to diamino-BP
so that within 4 hours diamino-BP was the only product present in the rumen.
Based on the metabolic data reviewed above, a metabolic pathway for
dinoseb was developed and is shown in Figure III-l.
III-6
-------
OH
NH,-C^~ ^C-CHCHjCHj
H"*C.v.
NHj
2-(2-butyrlc acidK 6-
dlamlnophenol
OH
X
C-CHCHaCHj
COOH
Olnoseb
ff-aec^utyl^.e-
dlnltfophanof)
9H
C-CHCHaCHj
H-C^-H
2-MC-
acet
6-nitrophend
OCH-(CHOHb-CHCOOH
NOj
2<2-butyrtcacldH,6-
dlnltrophenol
OH
C-CHCH2COOH
Ac,
2-(3*utyr1c acfdH, 6-
dlnitrophenol
2-sec-buty1-4^itro-
S^minophenylO
glucuronide
Figure III-l. Metabolic pathways of dinoseb in mammals based on reported
metabolites.
SOURCE: Adapted from Ernst and Bar (1964); Froslie and Karlog (1970); Bandal
and Casida (1972).
III-7
-------
D. EXCRETION
Bandal and Casida (1972) administered 8 to 10 #mo1/kg (1.9 to 2.4 mg/kg)
of "C ring-labeled DNBP (99% pure) by stomach tube to male albino rats
(180 g) and mice (20 g) and measured urinary and fecal excretion over a 72-
hour period. Animals were held in individual metabolism cages while receiving
food and water id libitum. Rats excreted approximately 65% of the radio-
activity in the urine and 25% in the feces within 72 hours (a total of
approximately 90% of the dose). Mice excreted 74% of the dose within
72 hours, with approximately equal quantities in the urine and the feces.
Gibson and Rao (1973) dosed nine nonpregnant female Swiss-Webster mice
with 32 mg/kg (orally) or 17.7 mg/kg (intraperitoneally) of 14C-labeled
dinoseb. Urine and feces were collected over the subsequent 64 hours, and
excretion was expressed as a cumulative percentage of the administered dose.
In a comparable study, the common bile duct of treated female mice was
cannulated, and bile was collected in tared scintillation vials. The bile was
weighed, solubilized, and counted for radioactivity. The results (shown in
Table III-3) indicated that in mice, orally ingested dinoseb is excreted in
both urine (26%) and feces (30%), with relatively lower levels found in the
bile (1.4% at 8 hours). Roughly similar values were observed in urine samples
after intraperitoneal dosing, but higher levels were observed in feces (41%)
and bile (10%).
Gibson and Rao (1973) measured the kinetics of clearance of 14C-dinoseb
from five female Swiss-Webster mice following a single oral dose of 32 mg/kg.
Excretion was first order, with a rate constant of 0.02 hour"1. This
corresponds to a t,/2 of 34.6 hours. Excretion was more rapid after a single
intraperitoneal dose of 7.7 mg/kg (rate constant » 0.09 hours"1, t,
7.7 hours).
'in
St. John et al. (1965) conducted a feeding experiment with four catheter-
ized Holstein cows receiving dinoseb in the grain feed at 5 ppm for 3 days.
No dinoseb residues were found in the milk. The levels of dinoseb in the
urine on days 1 through 6 after dosing were, respectively, 0.17, 0.05,
III-8
-------
Table III-3. Excretion of 14C-Dinoseb by Female Mice Following Oral or
Intraperitoneal Administration
Mean cumulative
Time after
administration
(hr)
0.5
1
2
4
8
16
32
64
Bile
Oral
(32 mg/kg)
0.1+0
0.4+0.1
0.6+0.3
0.9+0.4
1.4+0.6
_c
-
:
1p
(17.7 rag/kg)
0.2+0.1
0.6+0.1
1.4+0.4 .
3.9+0.6
9.6+1.4*
-
-
-
Oral
(32 rag/kg)
-
0.8+0.2
1.9+0.4
3.2+0.4
6.8+1.4
-14.4+2.0
23.2+3.5
26.3+3.3
excretion"
Urine
IP
(17.7 mg/kg)
1.4+0.2
3.9+0.1
7.0+0.1
13.4+1.3
22.1+2.1
26.3+1.9
28.2+2.5
Oral
(32 mg/kg)
-
-
-
-
0.5+0
4.3+1.1
9.7+3.7
30.4+7.5
Feces
(17.7 mg/kg)
- .-
-
-
-
3.3+0.9
11. 1+1.1*
- 28.7+4.8"
40.8+6.5
'Data are expressed as percentage of administered radioactivity. All values are
means for groups of three mice + SEM.
*p <0.05 (oral versus Intraperitoneal).
cNo data provided.
SOURCE: Adapted from Gibson and Rao (1973).
III-9
-------
0.18, 0.14, 0.09, and 0.46 ppm. Urinary excretion on day 6 represented 3.5%
of the administered intact compound as no conjugate formation was detected.
E. BIOACCUMULATION AND RETENTION
No studies were located that provided data on tissue or body levels of
dinoseb following long-term oral exposure.
F. SUMMARY
About 25 to 37% of an oral dose of dinoseb is excreted in the feces.
Approximately 40% of an intraperitoneal dose of dinoseb is excreted in the
feces, suggesting that fecal excretion results primarily from secretion of
absorbed dinoseb into the intestines via the bile. On this basis, absorption
may be estimated to be essentially complete. A maximum of approximately 7% of
the administered dose was found in the urine of monkeys following percutaneous
administration of dinoseb. Absorbed dinoseb is distributed to all tissues of
the mouse; however, brain and embryonic levels in pregnant mice never exceeded
2.5% of the maternal plasma level. Dinoseb is extensively metabolized by
several pathways in animals: (1) one or both of the nitro groups can be
reduced to the amine, Which may then be acetylated; (2) the terminal methyl
groups of the side chain can be oxidized to carboxyl groups; and (3) the
compound or its metabolites may be conjugated, primarily as glucuronides.
Although several metabolites have been identified, a greater number have been
detected but not yet characterized. Dinoseb and its metabolites are excreted
in both the urine and feces, with low amounts present in the bile. Excretion
was monophasic following a single oral dose, with a t,,z of about 35 hours in
mice. A t1/2 of about 8 hours was observed following an intraperitoneal dose.
111-10
-------
IV. HUMAN EXPOSURE
This section will be provided by the Science and Technology Branch, ODW.
IV-1
-------
-------
V.. HEALTH EFFECTS IN ANIMALS
A. SHORT-TERM EXPOSURE
1. Lethality
Acute oral lethality data for dinoseb are summarized in Table V-l.
Estimates of oral LD50 values in mice, rats, guinea pigs, and rabbits range
from 14 to 114 mg/kg. In a study by the Dow Chemical Company, it was reported
that the oral LDSO of dinoseb (purity of 96.8%) in rats was 58.29 mg/kg
(Industrial Biotest Laboratories, Inc., 1966).
Biggs et al. (1964), using dinoseb (purity of 18.5%) as a reference
compound, determined the oral LDSO to be 39, 29, 26, and 50 mg/kg in rats,
mice, guinea pigs, and rabbits, respectively.
The LDM values in rats and mice were reported in two studies by the Oow
Chemical Company (Mastri, 1970; Wazeter and Long, 1968). In the study with
rats, doses of 23.41 to 79.01 mg/kg (purity of 96.8%) were administerd orally
to two animals of each sex per dose group, and the LDSO was determined to be
79 mg/kg. In the study with mice, doses of 14.7 to 68.1 mg/kg (purity not
given) were administered orally to five male mice per dose group, and the LDSO
was determined to be 41.4 mg/kg; 0/5 mice died at the 31.6-mg/kg dose level,
and 4/5 mice died at the 46.4-mg/kg dose level. In additional studies by Rowe
et al. (1966a), dinoseb (purity not given) was administered by intubation, and
an LDM of 40 mg/kg (32 to 50 mg/kg) was calculated for rats; 25 mg/kg (20 to
31 mg/kg) for guinea pigs; and 26 mg/kg (18 to 37 mg/kg) for chicks. The dose
levels tested ranged from 5 to 60 mg/kg.
In a study conducted at Biochemical Research Laboratories, the LDSO in
rats was reported to be 114 mg/kg (Rowe et al., 1966b). Doses tested were
from 89 to 146 mg/kg (purity not given). In the same report, the LDSO is
reported to be 88 mg/kg (80 to 97 mg/kg) for guinea pigs and 70 mg/kg for
chicks (48 to 103 mg/kg). These values were higher than those of other
researchers.
V-l
-------
Table V-l. Acute Oral Toxicity of Dinoseb
Species
Mouse
Mouse
Mouse
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Rabbit
Sheep
Cattle
Chick
Chick
Chick
Sex
M
__»
M
M
M/F
--
M/F
M
M/F
. M/F
F
..
M/F
M/F
--
M/F
F
M
M/F
M/F
Dose
(mg/kg)
20-40
--
14-68
25-40
35 -
60
._
--
89-146
23-79
32-50
20-40
..
20-31
80-97
--
45
15 g
total
40-80
18-37
48-103
LDM (mg/kg)
(LD or No.
deaths/total
tested)
LD» ...
29
41
LDM
LD
LD
39
58
114
58
40
LDSO
26
25
88
50
2/4
0/1
LD.
26
70
Reference
Bough et al. (1965)
Biggs et al. (1964)
Wazeter and Long (1968)
Bough et al. (1965)
Ernst (1968)
Spencer et al . (1948)
Biggs et al . (1964)
Industrial Biotest
Laboratories Inc. (1966)
Rowe et al . (1966b)
Mastri (1970)
Rowe et al . (1966a)
Bough et al. (1965)
Biggs et al . (1964)
Rowe et al. (1966a)
Rowe et al. (1966b)
Biggs et al . (1964)
Froslie (1976)
Froslie and Karl og (1970)
Bough et al. (1965)
Rowe et al. (1966a)
Rowe et al. (1966b)
"Data not provided.
V-2
-------
Bough et al. (1965) reported on the acute oral toxicity of dinoseb
(purity 99%) In several animal species. Symptoms of poisoning in guinea pigs
included prostration, rapid respiration, and convulsions immediately preceding
death. Blood levels of DNBP in 10 female guinea pigs receiving 40 mg/kg
increased from 0 mg/100 ml to about 8.4 mg/100 ml at the time of death, even
though death occurred from 1.7 to 3.0 hours after dosing. Spencer et al.
(1948) conducted acute oral toxicity studies.of DNBP (purity of 99.1%) in rats
and reported that death occurred 1 or 2 hours after feeding or not at all.
The authors suggested that deaths were due to the pyretic effects of the
chemical. The authors report the "survival dose" (the largest dose that all
animals survived) as 5 mg/kg and the "lethal dose" (the smallest dose causing
death of all animals) as 60 mg/kg.
Palmer (1964) studied the toxicity of an alkanolamine salt of dinoseb
(purity not given) in 1- to 2-year-old Delaine-merino sheep, sex not speci-
fied. One sheep fed two daily doses of 100 mg dinoseb as the alkanolamine
salt died without preliminary signs of toxicity. A second animal received
four daily doses of 50 mg/kg/day by gavage and was also found dead without
premonitory signs. Necropsy findings included gastroenteritis, nephritis,
hepatitis, evidence of anemia (indicated by an enlarged spleen), areas of
hemorrhage, and edema of the heart.
The effect of ambient temperature on lethality was studied by Preache and
Gibson (1975b). Swiss-Webster female mice administered dinoseb (purity not
given) intraperitoneally were maintained at a high environmental temperature
(32°C) for 24 hours or at a low temperature (0 to 60°C) for 1.5 to 4 hours.
The LDSO at 32°C was 20.2 as compared to 14.1 mg/kg for the mice maintained at
0 to 6°C for 1.5 hours. The LDSO values after 1.5 and 4 hours at 0 to 6°C
were comparable.
The acute intravenous LD50 values for dinoseb in rats and mice were
reported to be 8 and 9 mg/kg, respectively (Biggs et al., 1964).
Acute dermal lethality data for dinoseb are summarized in Table V-2.
Estimates of dermal LDSO values in rats range from 67 to 134 mg/kg. Roughly
V-3
-------
Table V-2.. Acute Dermal Toxicity of Dinoseb
"""""^^^MHI
Species
Mouse
Rat
Guinea
pig
Rabbit
Rabbit
-^^ ^
Dose
Sex (mg/kg)
N 100
M 500
M 67-134
100
150
200
300
_
10
20
40
50
20
10
Application
site
Abdomen
Abdomen
Back
Abdomen
Abdomen
Abdomen
Abdomen
Abdomen
Back
Back
Back.
Skin
Skin
Skin
LDSO or
. No. deaths/
total tested
2/10
9/10
^«"i .^M^^^
Reference
Bough et al.
Bough et al.
^ ^«»t===]i===
(1965)
(1965)
LDSO Noakes and Sanderson
(1969)
0/5
1/5
4/5
5/5
3/3
0/4
4/4
4/4
2/2
0/2
0/2
Spencer et al
Spencer et al
Spencer et al
Spencer et al
Spencer et al
Bough et al.
Bough et al.
Bough et al .
Wolf (1959)
Wolf (1959)
Wolf (1959)
. (1948)'
. (1948)
. (1948)
. (1948)
. (1948)
(1965)
(1965)
(1965)
'Death occurred following three to eight applications of 3% alcoholic solution of
dinoseb. Total dose not specified.
V-4
-------
similar results for the dermal toxicity of dinoseb have been reported in mice
and guinea pigs, but rabbits may be more sensitive to the chemical (Spencer
et al., 1948; Bough et al., 1965; Noakes and Sanderson, 1969). In a study by
the Dow Chemical Company, the acute dermal LDM for guinea pigs was reported
to be in the range of 100 to 500 mg/kg dinoseb (purity of 99.1 to 99.7%). The
survival dose was 100 mg/kg. No other information was provided.
2. Other Acute Effects
Dandliker et al. (1980) reported the effect of a single oral dose of
dinoseb (about 20 mg/kg) (purity not given), administered by intubation, on
the immune response of inbred male hamsters, strain LHC/LAK, age 5-to 8 weeks
and weighing about 100 g. The dose was described as one-half the LD50 re-
ported by Thompson (1976), which was 37 to 50 mg/kg. Dinoseb markedly
depressed the cellular immune response as measured by two methods: (1) visual
elevation of the intensity of inflammation and swelling of an antigen-injected
footpad as compared with the centralateral footpad treated with buffer alone;
and (2) differential temperature measurement between the antigen-injected
footpad and the control footpad. Dinoseb also depressed the humoral immune
response, as measured by fluorescence polarization measurements after adding
fluorescein to an Ig preparation from serum.' The authors point out that the
actions of dinoseb were remarkably long lasting (remaining 49 days after
dosing) and suggest that the effect is probably the result of a decreased
level of antibodies rather than of any change in the type of antibodies
produced.
Froslie and Karlog (1970) reported that a single 15-g dose of DNBP
(purity not given) administered into the rumen of two cows produced toxic
effects, including increased pulse rate and total anorexia. A reddish-brown
urine was observed. At 2 to 4 hours after ingesting the herbicide, the cattle
had methemoglobin blood levels of 30 to 40%. Within 2 weeks, however, the
animals appeared to recover. A methemoglobin concentration of 10% could still
be measured after 10 days, and hemolysis persisted for several days.
V-5
-------
Froslie (1974) reported a study of acute DNBP (purity not given) toxicity
in sheep. Seven sheep received a single dose at 45 mg/kg administered by tube
into the rumen. The dinoseb was dissolved in approximately 200 ml of a 33%
solution of acetone in water. All animals showed hemolysis of red blood cells
and methemoglobinemia formation. The acute phase of toxicity (lasting from 6
to 8 hours) was characterized by dyspnea, hyperthermia, methemoglobinemia, and
hemoconcentration. After 1 to 2 days, hemoglpbinemia and hemoglobinuria were
the predominating clinical signs. The methemoglobinemia lasted for 2 to 3
days with maximum methemoglobin values of 5 to 8 g/100 ml. Liver and kidney
dysfunction and a significant reduction in plasma proteins occurred during
this acute phase. Glutathione levels in the red blood cells were also
markedly decreased. Postmortem lesions consisted of moderate congestion and
degenerative changes in the liver and kidney. One animal showed the
discoloration of methemoglobinemia, while other animals that died during a
hemolytic crisis were icteric, with extensive degenerative changes in the
liver and kidneys. Two animals died and one was euthanized during the first 4
days of the study; all others survived. The author concluded that the effects
of DNBP include both an initial and a delayed phase. The initial effect is
partly related to the unmetabolized dinitrophenol, which produces dyspnea and
hyperthermia. A major factor in this stage, however, is the formation of
diamino metabolites in the rumen, which results in methemoglobinemia,
hypoproteinemia, and hemoconcentration. According to the author, the diamino
metabolites also contribute to the later stages of poisoning, which include
lysis of red blood cells.
Spencer et al. (1948) investigated the potential of dinoseb (purity not
given) to produce cataracts in White Pekin ducklings. The experiment was
based on the knowledge that 2,4-dinitrophenol produces cataracts in humans and
that ducklings and chicks appear to be suitable laboratory animals for such
studies. Ducklings that received 0.25% dinoseb in the diet died within 3 days
but had no cataracts. Animals receiving 0.1% in the diet died within 4 days,
with one animal showing cataracts. Half of the animals ingesting dietary
levels of 0.03% died within 5 days. At this exposure, cataracts were observed
in one duckling on the fifth day and in another on the eighth day, at which
V-6
-------
time the birds were accidentally killed. This study demonstrates the ability
of dinoseb to produce cataracts in experimental animals.
Spencer et al. (1948) reported that of 10 albino rats (Breeding and
Laboratory Institute, Brooklyn) fed a diet delivering dinoseb at 13.5 mg/kg/-
day (99.1%), 4 died between days 5 and 13. The remaining six animals were
sacrificed on day 21. They showed marked emaciation, an empty gastro-
intestinal tract, and a blood urea nitrogen of 55 mg% (controls: 19.4 mg%).
Microscopic examination of tissues revealed slight degenerative changes in the
renal tubules and slight cloudy swelling of the liver, but no appreciable
changes in lung, heart, spleen, adrenal, pancreas, or testes.
Pawlowski (1970) studied the central nervous system (CNS) effects of
dinoseb (purity not given) in the Wistar rat. The animals were dosed intra-
peritoneally once each day for 5 days. Dose levels of 2 and 8 mg/kg/day were
without effect. Doses of 12 or 16 mg/kg/day intensified inhibitory and
excitatory activities in the brain, resulting in shorter periods of time
needed for the development of escape reaction, increased infrequency of
positive conditioned reactions, and more efficient differentiation between
stimuli. Doses of 20 or 24 mg/kg/day provoked the inhibition of conditioned
reflex activity.
B. LONG-TERM EXPOSURE
1. Subchronic Effects
Hall et al. (1978) reported on the toxic effects of dinoseb (purity of
80%) administered in the diet to 35- to 38-day-old Sherman rats for 60 days.
Groups of 14 rats of each sex were fed a diet fortified with technical grade
dinoseb at 0, 50, 100, 150, 200, 300, 400, or 500 ppm. All animals receiving
dietary levels of 400 and 500 ppm died within 3 weeks. Of the animals
receiving 300 ppm, 14% died within 21 days, and this group was not continued
on the diet. Growth was depressed at all lower dietary levels (50 to
200 ppm). Organ weights (liver, spleen, heart, lung, brain) decreased, and
V-7
-------
organ-to-body weight ratios Increased. Blood alkaline phosphatase and alanine
aminotransferase activities and potassium and urea nitrogen contents were
significantly increased, and lactic dehydrogenase and cholinesterase activi-
ties were decreased. Tissue residue levels were dose related in the following
order: blood>feces>urine>adipose>brain>liver. Discrimination learning was not
affected, and locomotor activity was increased at 200 ppm. Diffuse tubular
atrophy of the testes was observed, particularly in animals receiving 200 ppm.
Assuming that 1 ppm in the diet equates to a dose of approximately 0.05 mg/-
kg/day, the dietary level of 50 ppm equals 2.5 mg/kg/day. Based upon the
organ-to-body weights and hematological changes, no NOAEL was obtained, and
2.5 mg/kg/day was established as a LOAEL.
* '' m
Spencer et al. (1948) conducted a 6-month dietary study with white male
rats {Breeding and Laboratory Institute, Brooklyn) fed dinoseb (99.1% pure)
mixed in the diet. Food and water were available ad libitum. Thirty rats
served as controls; three groups of 20 rats were administered dinoseb at
dietary levels of 1, 3.5, 2.7, or 5.4 mg/kg/day; and 10 rats received
13.5 mg/kg/day.. Nonpalatability was observed, although the actual food intake
was not reported. Four rats that received 13.5 mg/kg/day died within 13 days;
the six survivors appeared markedly emaciated and were sacrificed on day 21.
The body weights of the animals receiving 5.4 mg/kg/day were 3 to 8% lower
than those of the control animals during the 6-month study period (p <0.05).
No other discernible toxic effects were noted in these animals during the
study. No effects on erythrocyte count, hemoglobin concentration, leukocyte
count, and differential count were observed. The average blood urea nitrogen
was 20.3 mg/100 ml compared with 17.5 mg/100 mL for the controls. Organ
weights were comparable to those of controls, except for an increase in liver
weight (p <0.01) in rats fed 5.4 mg/kg/day of dinoseb. Gross and microscopic
examination of tissues failed to reveal any appreciable changes. Animals at
the two lower dosage levels (2.7 or 1.35 mg/kg/day) had growth curves that
were comparable to those of the controls; blood urea nitrogen levels, organ
weights, and histopathology were also similar to the control group. Thus,
2.7 mg/kg/day represents a NOAEL for 6-month dietary exposure in rats.
V-8
-------
In a study conducted by McCollister et al. (1967), groups of four male
and four female beagle dogs per dose were fed diets containing 0.005, 0.01,
0.02, or 0.03% dinoseb (purity of 97.5%) for 91 days. Dogs receiving 0.005 or
0.01% did not exhibit adverse effects. Females receiving 0.02 and 0.03%
showed slight growth retardation, increased average weights of liver, mural
endocarditis, and microscopic heart changes. Dogs removed from these test
diets after 91 days and given half their basic ration for an additional
37 days gained weight, and their average liver-to-body weight ratios in-
creased; no histopathological changes were seen. The NOAEL was established as
4 mg/kg/day, based upon the lack of effects at 0.01% (100 ppm). The dose
conversion was based on food consumption data.
2. Chronic Effects
Two chronic toxicity studies have been reported, one with rats and the
other with mice. Hazleton (1977) conducted a 2-year feeding study with groups
of 60 albino rats/sex (Charles River CD) at dose levels of 0, 1, 3, and
10 mg/kg/day (purity not given). Hunched appearance and staining of the fur
were noted more often in the dosed animals when compared to controls.
Polypnea was noted in all treated animals, particularly females, during the
first year of the study. Mean body weight gains of males receiving the mid
and high doses and females receiving all doses were slightly to moderately
lower than those of controls during the first year of the study; the decrease
was statistically significant. At study termination, the mean body weights
were still lower than those of controls, but the decreases were not statis-
tically significant. There were no compound-related effects on survival, food
consumption, hematology, clinical chemistry, and urinalysis. Palpable nodules
and tissue masses were first noted by week 34 and were more frequently seen in
females than males. Gross pathology showed lung abnormalities and liver
discolorations in dosed and control animals. There were no effects on mean
organ weights between control and dosed animals. However, a significant
(p " 0.05) decrease in mean thyroid weight was observed at all dose levels in
male rats. A dose-related trend in decreased thyroid weights was also
observed. No histopathological changes were detected, but tissues for only
V-9
-------
10 animals per sex from the control and high-dose groups and the liver,
kidneys, and lesions from the low- and mid-dose rats were examined at interim
sacrifice on week 52 and at final sacrifice on week 104. Based on decreased
thyroid weights, a NOAEL was not established for this study, and 1 mg/kg/day
was designated as a LOAEL.
In a chronic feeding study with mice, groups of 70 male and 70 female CD-
1 mice were administered 0, 1, 3, and 10 mg/kg/day of technical grade dinoseb
(purity of 98%) in the diet for 100 weeks (Brown, 1981). Beginning in week 10
of the study and lasting throughout, body weight gain was reduced in the mid-
and high-dose females only. Low-dose females and all males were unaffected.
Food consumption, hematology, and urinalysis did not reveal any treatment-
related changes. A small number of high-dose males showed very high
fluctuations in plasma alkaline phosphatase, while most of the animals in the
group were within normal range. The group differences were not statistically
significant. Lenticular opacities were observed at the 3- and 10-mg/kg/day
dose levels, but animals receiving the low dose were not examined. Cystic
endometrial hyperplasia and atrophy were observed in females, and.hypo-
spermatogenesis and degeneration were seen in the testes of males receiving 1,
3, and 10 mg/kg/day. Thus, no NOAEL was identified, and the LOAEL for this
study was 1 mg/kg/day.
C. DEVELOPMENTAL/REPRODUCTIVE EFFECTS
1. Developmental Effects .
a. Oral
Gibson (1973) reported on the teratogenic effects of dinoseb (purity not
given) in Swiss-Webster mice. The compound was administered, by gastric
intubation, at doses of 20, 32, or 50 mg/kg/day in aqueous solution to groups
of pregnant Swiss-Webster mice on days 8 to 16, 10 to 12, or 14 to 16 of
gestation. Significant increases of supernumerary ribs were found when
20 mg/kg dinoseb was administered throughout organogenesis (8 to 16 days). At
V-10
-------
32 mg/kg/day, a slight, but significant, reduction in fetal crown-rump
distance (2.4 cm compared with 2.6 cm in controls) was observed along with a
significant increase of supernumerary ribs and vertebrae, and absent or
unossified sternebrae. Doses of 50 mg/kg killed approximately 75% of the dams
but produced no effects on fetal survival or size. Skeletal anomalies were
not observed; however, only two litters were available for examination owing
to maternal toxicity. The author concluded .that the NOAEL for dinoseb
administered orally during organogenesis was 20 mg/kg/day, even though there
was a statistically significant increase in the incidence of supernumerary
ribs. It was pointed out that the positive findings were present only at dose
levels associated with some maternal lethality.
Chernoff and Kavlock (1983) studied the maternal and perinatal effects of
dinoseb (purity not given) administered orally at 15 mg/kg/day to pregnant CD-
1 mice. The chemical was given on gestation days 8 through 12. No effect was
observed on maternal weight, number of surviving pups, or pup weights 1 and 3
days postpartum.
In another study, Kavlock et al. (1985) assessed the effects of acute
maternal toxicity upon fetal development. Pregnant CD-I mice were placed in
groups of 15, 20, and 40, and orally administered single doses of dinoseb
(purity of 97%) at 0, 26, and 33 rag/kg, respectively, on day 8 of gestation.
The animals were sacrificed on day 18 of gestation, and the uteruses were
removed and weighed. The fetuses were removed from the uterus, weighed, and
examined for any gross malformations. The only significant fetal effect was
an increase in the incidence of supernumerary lumbar ribs. This effect was
inversely related to maternal weight gain. Other isolated malformations
include encephalocele, exencephaly, miscellaneous cranial defects, and an
umbilical hernia.
Spencer and Sing (1982) reported the effect of dinoseb (purity of 95%) on
pregnant Sprague-Dawley rats. Dinoseb was added to the diet from days 6
through 15 of gestation. Decreased maternal body weights, ataxia, lethargy,
decreased levels of placental protein and glycogen, and a significant
V-ll
-------
reduction In embryo survival were found at doses of 8.60, 9.38, 9.49, and
10.86 mg/kg. Consumption of 9.23 mg/kg djnoseb resulted In decreased maternal
weight and a significant decrease in pup survival at birth. A dose of 6.9
mg/kg/day did not result in maternal weight loss or other toxic signs;
however, there was a nonsignificant decrease in fetal survival. At higher
doses, reduction in fetal survival reached statistical significance. The
effect of dinoseb on pregnancy performance is shown in Table V-3. In this
study, the NOAEL was found to be 3.26 mg/kg/day, and the LOAEL was found to be
6.9 mg/kg/day.
In a study with pregnant Wistar rats, dinoseb (purity of 96.1%) was
administered by gavage to groups of 25 mated rats at doses of 0, 1> 3, or
10 mg/kg/day on days 6 through 15 of gestation (Becker, 1986a). Decreased
total body weight gains (not significant) were noted in dams receiving
10 mg/kg/day. Reduced ossification corresponded with the significantly reduced
fetal body weights (p <0.05) at the high dose and were considered to be an
effect of the maternal toxicity. There was an increase in the incidence of
bilateral supernumerary ribs at the high-dose level. The developmental NOAEL
was identified as 3 mg/kg/day.
Becker (1986b) administered dinoseb (purity of 98.1%), by gavage, to
groups of 16 mated chinchilla rabbits at doses of 0, 1, 3, or 10 mg/kg/day
from days 6 though 18 of gestation. No maternal toxicity was noted in any
group. Increased incidences of external, visceral, and skeletal malformations
seen in the high-dose group were considered to be compound-related and
included anomalies in 40 fetuses (32.8%) in 11 of 16 litters. Multiple
anomalies were noted in 26 fetuses (21.3%). Compound-related malformations
were predominantly microphthalmia, anophthalmia, and internal hydrocephaly.
Neural tube defects such as dyscrania associated with hydrocephaly, scoliosis,
kyphosis, dysmorphogenesis of caudal and sacral vertebrae, and encephalocele
were noted in approximately 31% of the litters from dams receiving 10 mg/kg/-
day. Oinoseb was considered to be teratogenic at levels of 10 mg/kg/day. The
developmental NOAEL and LOAEL were 3 and 10 mg/kg/day, respectively. The
maternal NOAEL was 10 mg/kg/day.
V-12
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Table V-3. Effect of Dinoseb on Pregnancy Performance in Rats
Dietary Intake of Number
treatment* dlnoseb of
(ppm) (ing/kg/day) Utters
0
50
100
150
ZOO
250
300
350
~
3.26 t
6.90 t
9.23 i
10.86 t
9.38 i
9.49 t
8.60 i
0.09
0.20
1.02
1.33
2.05 -
1.46
1.57
6
6
6
6
6
6
6
6
Implantations
at day 6
per dam
12.5 i
. 11.7 t
13.7 t
14.3 t
13.2 t
12.3 t
14.8 t
12.2 t
1.4"
1.9
1.1
0.7
1.4
0.7
2.1
0.4
Number of
conceptions
at day 12
12.5 t 1.4
11.7 t 1.9
13.7 i 1.1
14.3 t 0.7
9.7 t 1.1
7.3 i 2.6
4.9 t 3.5
0.0 t 0.0
Percent
embryo
survival
per litter
at day 12b
100 t
100 t
100 t
100 t
75 i
56 t
33 t
00 t
00.0
00.0
00.0
00.0
9.0*
22. 3e
21.0*
00.0
Percent pup
survival
per litter
at birth0
80.12 ±
83.31 ±
63.09 t
45.9 t
53.06 t
16.34 ±
10.81 t
0.00 t
7.59
12.56
6.05
11.568
9.20*
12.32*
5.56*
0.00
Fetal birth
weight per
litter (g)
7.20 t 0.30
7.13 t 0.27
6.78 t 0.14
6.43 1 0.18e
aD1noseb administered from day 6 though 15 of pregnancy.
"Percent embryo survival: the ratio of the number of surviving embryos per litter at day 12 to that at
day 6, as expressed in percentage.
'Percent pup survival: the ratio of the number of live pups at birth to the number of implantation sites counted
at day 6, as expressed in percentage.
All results expressed as mean ± SE.
'Significantly different from the control, using Student's test (p
-------
Gray and'Kavlock (1984) orally dosed pregnant CD-I mice with dinoseb
(purity of 97%) and determined development. Pregnant CD-I mice were adminis-
tered oral doses of dinoseb at levels of 0 and 5 mg/kg daily during days 8 to
12 of gestation. At that time, the pups were weighed, counted, and, along
with the dams, randomly separated and housed in litters of six animals. At
30 days of age, the pups were weaned, counted, weighed, and housed for
breeding purposes. During the 250-day study period, the mice were observed
for any changes or lingering neonatal effects. Pups born during the study
were counted and the length of gestation recorded. At 250 days of age, the
males were sacrificed and necropsied. No significant effects on pup survival
and weight, weaning survival and weight, viability, body and organ weight, or
gross pathology were noted. -
b. Intraoeritoneal
Gibson (1973) also administered intraperitoneal doses (purity not given)
of 0, 10, 12.5, 15.8, 17.7, 18.8, and 20.0 mg/kg/day to Swiss-Webster mice on
gestation days 10 to 12. Additional mice received 0, 12.5, or 17.7 mg/kg/day
on days 14 to 16 or 5 mg/kg/day on days 8 to 16. Doses of 15.8 mg/kg or below
were not maternally toxic, whereas hyperthermia and some lethality were noted
at 17.7 and 18.8 mg/kg. None of the dams survived at 20 mg/kg. Subtoxic
doses of 10 to 15.8 mg/kg had no developmental effects when given during days
10 to 12; however, increased resorption rates and reduced fetal weights were
significant for dams given 12.5 mg/kg on days 14 to 16. Fetuses from dams
dosed with 17.7 mg/kg/day on days 10 to 12 of gestation .had reduced weights
and a variety of gross anomalies (oligodactyly, imperforate anus, acaudia,
microcaudia, and amelia), soft tissue anomalies (internal hydrocephalus,
hydronephrosis), and skeletal anomalies (fused ribs, missing ribs, fused and
missing sternebrae, fused, unossified, or absent vertebrae, and absent or
unossified long bones). Fetuses from the 18.8-mg/kg group had significantly
reduced weights and lengths and increased incidences of fused vertebrae and
fused ribs when compared to controls. An intraperitoneal dose of 5 mg/kg/day
given throughout organogenesis (days 8 to 16) produced no developmental
effects.
V-14
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Preache and Gibson (19755) studied the effect of drugs that alter hepatic
drug-metabolizing activity on the fetal toxicity of dinoseb (purity not
given). Swiss-Webster mice were treated intraperitoneally with dinoseb at
doses of 0, 14.1, or 15.8 mg/kg/day on days 10 to 12 of gestation. Subgroups
of each set of dosed mice were deprived of food for 0, 24, or 48 hours from
the ninth day of gestation. In a second study, two groups of pregnant mice
were given single injections of either 17.7 mg/kg on day 11 of gestation or
18.8 mg/kg on day 12 of gestation. For approximately half of the mice in each
group of the second study, dinoseb administration was preceded by treatment
with 50 mg/kg phenobarbital twice each day for 3 days; the remaining mice were
not pretreated. Two other groups received 15.8 or 17.7 mg/kg dinoseb 1 hour
after treatment with 32 mg/kg of SKF-525A on day 12 of gestation. A third
group served as the untreated control. On the 19th day of gestation, fetuses
were removed by cesarean section and were weighed and examined for external
anomalies. Half of the fetuses of each litter were fixed in Bouin's solution
and examined for soft tissue anomalies. The remaining fetuses were stained
with alizarin red S and examined for skeletal defects.
The results of this study indicated that SKF-525A potentiated and pheno-
barbital inhibited the resorptions and reductions in fetal body weight induced
by dinoseb. Dinoseb-induced external, soft tissue, and skeletal anomalies
were increased by 24-hour food deprivation and SKF-525A pretreatment, and
48-hour food deprivation had minimal adverse effects on fetal weight and
ossification of small bones. In general, the action of phenobarbital pro-
tected against dinoseb teratogenicity. Disposition of radiolabeled dinoseb
(15.8 mg/kg) was also examined in adult female mice following each pretreat-
ment. Food deprivation for 24 hours slowed and phenobarbital pretreatment
hastened the disappearance of dinoseb from the plasma. Food deprivation for
48 hours and SKF-525A pretreatment did not affect the disappearance of dinoseb
from the plasma but did increase and decrease, respectively, its disappearance
from the liver. The authors suggested that the alterations in dinoseb-induced
embryotoxicity and teratogenicity produced by the pretreatments were related
V-15
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to an alteration In the rate of oxidative metabolism and clearance of dinoseb
from the mother {Preache and Gibson, 1975b).
McCormack et al. (1980) reported a study of postnatal morphology and
functional capacity of the kidney iii neonates of Sprague-Dawley rats injected
intraperitoneally with dinoseb (purity not given) at doses of 0, 6.3, 8.0,
9.0, 11.2, 12.5, or 15.8 mg/kg/day on days 10 to 12 of gestation. At 21 days,
some fetuses were'removed by cesarean section and examined, and samples were
selected for histological.examination. Histological examination was also
performed on selected tissues from offspring at day 1 or 42 postpartum. Renal
function of rats exposed to dinoseb prenatally was determined both in vivo and
in vitro. Transport capacity in the kidney was determined by measuring the
ability of tissue slices to accumulate p-aminohippuric acid or N-methyl-
nicotinamide. Postnatal renal function was also assessed in the 42-day-old
rats by measuring inulin and p-aminohippuric acid clearance, blood urea
nitrogen, and maximal urine osmolarity. Pregnant rats administered dinoseb at
11.2, 12.5, or 15.8 mg/kg/day all died within 1 week of treatment. At the
dose of 9.0 mg/kg, the mortality rate of pregnant rats was 20%. At 8.0 mg/kg
or less, no deaths occurred. Fetal weight was decreased by dinoseb at doses
of 8.0 or 9.0 mg/kg/day. Fetal length was also reduced at the dose of
9.0 mg/kg. Postpartum studies indicated that pups exposed in utero weighed
less than controls at days 1 and 7 postpartum but were not different from
controls at 42 days of age. Livers from near-term fetuses of mothers treated
with 8.0 to 9.0 mg/kg dinoseb on days 10 to 12 had many vacuolated cells.
This effect persisted even through 42 days postpartum, along with the presence
of necrotic cells. The nucleus was absent from soma cells and was pyknotic or
karyorrhectic in others.
Kidney-to-body weight ratios were not affected by dinoseb treatment.
However, approximately 40% of the near-term fetuses from dams treated with 8
or 9 mg/kg/day had dilated renal pelves and/or ureters when examined grossly.
Histological examination revealed dilation of renal pelves and tubules. The
transitional epithelium was vacuolated in ureters from near-term and 1-day-old
rats treated with dinoseb in utero. The incidence and severity of kidney
V-16
-------
lesions decreased with age of offspring. At 42 days postpartum, gross
examination of kidneys and ureters did not reveal any dilated ureters, and
only 3 of 28 animals at the prenatal dose of 9 mg/kg/day had dilated renal
pelves. No microscopic differences were noted between ureters and kidneys of
dinoseb-treated and untreated rats at 42 days of age. Other renal parameters
measured in offspring were not affected by dinoseb treatment. In this study,
the NOAEL for the pregnant females exposed on days 10 to 12 of gestation was
6.4 mg/kg. The authors suggest that the toxicity of dinoseb in pregnant female
mice is greater than that in rats.
c. Subcutaneous
In addition to the oral and intraperitoneal studies, Gibson (1973) showed
that subcutaneous injections of 17.7 mg/kg/day (purity not given) during late
organogenesis (days 14 to 16 of gestation) or throughout organogenesis (days 8
to 16) produced overt maternal toxicity and decreased fetal survival and size
in mice. Statistically significant gross or soft tissue anomalies were
produced with doses of 17.7 mg/kg/day on days 14 to 16. An increased
incidence of skeletal anomalies occurred in the offspring of dams treated with
17.7 mg/kg/day on days 10 to 12 and 8 to 16, but not during late organogenesis
(days 14 to 16). A subcutaneous dose of 10 mg/kg/day revealed no effect at
any of the dosing periods.
d. Dermal
In a dermal developmental toxicity study conducted by Argus Research
Laboratories, Inc. (Hoberman, 1987), groups of 17 artificially inseminated New
Zealand White rabbits were percutaneously administered dinoseb (99% purity) at
doses of 0 (sham control), 1, 3, or 9 mg/kg/day on days 7 through 19 of
gestation. In addition, 12 artificially inseminated rabbits were percu-
taneously dosed with 18 mg/kg/day. These doses were based on a dermal range-
finding study in which nonpregnant female rabbits were given doses of 10, 25,
50, or 75 mg/kg daily for 3 days. After 2 days of dosing, however, a mor-
tality rate greater than 10% occurred in groups receiving 9 or 18 mg/kg/day.
V-17
-------
The dose level of remaining high-dose animals was then reduced to 9 mg/kg/day,
and four untreated animals were reassigned to the 9-mg/kg/day group and
received this dose level for the entire 13-day dosing period.
The animals were fitted with Elizabethan collars and exposed to dinoseb
for approximately 6 hours a day, after which test sites were rinsed with an
isopropanol water solution and blotted dry to remove the test material. The
skin at the application site was evaluated daily, prior to dosing, using the
Draize method to grade erythema, edema, and possible eschar formation.
Surviving animals were delivered by cesarean section on day 29 of gestation.
All dams, including those that died prior to study termination, .animals that
aborted, and sacrificed animals, were examined grossly, and a tera-tological
evaluation of their offspring was conducted. Visceral and skeletal examina-
tions were conducted on each fetus, and brains were examined using a single
cut at the level of the anterior fontanelle.
As previously stated, a significant increase {p <0.01) in mortality was
noted in the high-dose groups. Mortality rates of 71% {15 of 21 animals) and
88% (7 of 8 animals) occurred in rabbits receiving 9 and 9(18) mg/kg/day of
dinoseb. The author also considered the slight increase in mortality at
3 mg/kg (3 of 17 animals) to be due to dinoseb administration. Administration
of dinoseb resulted in slight to moderate dermal irritation. Measurement of
daily rectal temperatures indicated that body temperatures were elevated in
animals receiving dosages of dinoseb at 3 mg/kg/day or higher. Total body
weight gains for rabbits receiving dinoseb were slightly, increased compared to
those of controls (Table V-4). However, during the period of dose administra-
tion (gestational days 7 through 19), body weight gains were decreased for all
groups, although the dinoseb-treated animals appeared to be more severely
affected. These body weight decreases were probably due in part to the
wearing of Elizabethan collars.
Gross observations of dams given 9 and 9(18) mg/kg/day attributable to
dinoseb administration included the appearance of yellow musculature and
V-18
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Table V-4. Summary of Body Weight Changes In Pregnant Rabbits Percutaneously
Treated With Dinoseb During Days 7 to 19 of Gestation
Dosage level .
(mg/kg/day)
0
1
3
9
9(18)
Body
0
3.74 ± 0.37'
3.81 + 0.33
3.70 ± 0.31
3.74 ± 0.38
3.51 ± 0.32
weiahts (ka) at
7
3.85 ± 0.38
3.95 ± 0.33
3.83 + 0.31
3.88 ± 0.28
3.64 ± 0.36
aestational
19
3.73 ± 0.45
3.70 ± 0.42
3.57 ± 0.46
3.68 ± 0.33
4.03 ± 0.00
dav:
29
3.80 ± 0.44
3.90 ± 0.32
3.82 ± 0.42
4.00 ± 0.-23
4.20 ± 0.00
Total
weight
gain
0.06
0.09
0.12
0.26
0.69
Mean ± SD.
SOURCE: Hoberman (1987).
V-19
-------
subdermal tissue, and hemorrhaging of the brain, trachea, thymus, lungs, and
subdermis of the thorax and abdomen.
Reproductive parameters such as numbers of implantations, corpora lutea,
live fetuses per litter, and fetal body weights and sex ratios were comparable
between control and dosage groups. Mean number of resorptions appeared to be
increased (not statistically significant), and live litter size was decreased
for the group receiving 9 mg/kg/day. A summary of reproductive parameters is
presented in Table V-5.
Oose-related increases were observed in gross external, soft tissue, and
skeletal malformations in offspring of dams given dinoseb at levels of 3 mg/kg
or higher. Malformations attributed to dinoseb administration in the 3-, 9-,
and 9(18)-mg/kg/day dosage groups included hydrocephaly, ectopic eye bulge,
microphthalmia, anophthalmia, craniosynostosis (includes all malformations
related to fusion or incomplete development of the calvaria), and small eye
socket (Table V-6). In the most severely affected fetuses from the high-dose
groups, the anterior fontanelle,.frontals, parietals, and/or nasals were
fused, the zygomatics were short and/or flat, and the skull was abnormally
shaped and small with incomplete ossification of the nasals and hypoplasia of
the nasal portion of the eye sockets and communication of the sockets. Also
frequently noted in these fetuses were hemivertebrae; fused, asymmetric, or
unilaterally ossified centra; and short or absent tails. Less frequently
noted were macrophthalmia, meningocele, cleft lip and/or palate, protruding
tongue, and gastroschisis. Significant increases (p <0..01) in the incidence
of delayed ossification of the parietals was noted in fetuses from dams
treated percutaneously with 3 mg/kg or higher of dinoseb.
The NOAEL for both maternal and developmental toxicity for this study was
1 mg/kg.
V-20
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Table V-5. Summary of Embryo/Fetal Toxicity in Pregnant Rabbits Percutaneously
Treated With Dinoseb During Days 7 to 19 of Gestation
Dosaae level (ma/kq)
Parameter
No.
No.
(*;
No.
tested
pregnant
aborted
0
17
16(94.1)
2
1
17
16(94.1)
4
3
17
16(94.1)
2
9
21
17(81.8)
2
9(18)
8
8(100)
0
No. surviving
and pregnant 10 10 .11 3 1
No. live fetuses/
litter 6.5 + 2.8* 7.1 ± 3.1 8.3 + 2.3 3.3 + 2.1 7.0 + 0.0
No. dead fetuses 0 00 0 0
No. resorptions
Early 0.8 + 0.9* 0.1 + 0.3 0.5 + 0.7 4.0 + 3.0 0.8 + 0.8
Late 0.1+0.3* 0.1 + 0.3 0.3 + 0.6 0.0 + 0.0 0.0 + 0.0
fetal body
weight/litter (g) 42.2 + 8.51 39.3+7.4 37.2 ± 4.7 48.1+5.0
'Mean ± SD.
"Fetal body weights invertently not recorded (one dam affected).
V-21
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Table V-6. Incidences (%) of Litter and Fetal Malformations Found
in Rabbits Percutaneously Treated With Oinoseb During
Days 7 to 19 of Gestation
Dosaae level (ma/ka)
Tissue/
malformation
Skull;
Microcephaly
Litter
Fetal
Frontal s fused
Litter
Fetal
Eye sockets small
Litter
Fetal
Craniosynostosis
Litter
Fetal
Eves;
Microphthalmia
Litter
Fetal
Anophthalmia
Litter
Fetal
Bulge, depressed,
reduced, and/or
ectopic
Litter
Fetal
Brain;
Hydrocephaly
Litter
Fetal
1
0
0
0
0
0
0
0
0
1(10.0)
1(1.5)
0
0
1(10}
1(1.5)
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
2(18.2)
3(3.3)
1(9.1)
1(1-1)
2(18.2)
3(3.3)
0
0
1(9.1)
1(1«D
0
0
2(18.2)
2(2.2)
9
1(33.3)***
1(10.0)**
3(100)**
6(60)**
2(66.7)**
6(60.0)**
3(100)**
8(80.0)**
2(66.7)**
5(50.0)**
3(100)**
3(30.0)**
3(100)**
6(60.0)**
3(100)**
7(70.0)**
9(18)
1(100)**
6(85.7)**
1(100)**
6(85.7)**
1(100)**
6(85.7)
1(100)**
6(85.7)**
1(100)**
7(100)**
1(100)**
1(14.3)**
1(100)**
7(100)**
7(100)**
6(85.7)**
'Number affected (incidence).
**Significantly different from controls (p <0.01).
SOURCE: Hoberman (1987).
V-22
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2. Reproductive Effects
Groups of 25 male and female rats in each of three generations received
dinoseb (purity 98.4%) in the diet at dose levels of 0, 1, 3, and 10 rrg/kg/day
for 29 weeks (Irvine and Armitage, 1981). Both sexes at the high-dose level
shewed a lo/ver rate of body weight gain in all three generations. This effect
was inconsistent at lo/\er dose levels and across generations. Dinoseb at the
10-rrg/kg/day level elicited no effects on survival, fertility, or fecundity,
and no microscopic or macroscopic changes; however, the nirrbers of pups born
and pup weights at weaning were decreased at all dose levels and were
attributed to maternal toxicity {lo/\er maternal body weight). Findings at the
loner dose levels, including minor skeletal defects in the Fj, generation, were
considered incidental. A U3VEL of 1 mg/kg/day was identified.
An additional two generations were studied as a continuation of the above
three-generation study (i.e., five consecutive generations were
studied); the same doses were administered (Irvine and Armitage, 1981).
Reduced gains in high-dose parental body weights persisted in the fj and F4
generations. F4 litter sizes at birth were reduced at 10 nrg/kg, and $
preweaning pup weights were slightly reduced at 3 and 10rrg/kg. Na progeny
effects were noted for the F5 generation. A LCXVEL of 1 mg/kg/day was calcu-
lated.
Under et al. (1982) studied the effects of DVBP (purity of 97.3%)
feeding on spermatogenesis in male rats. Technical grade dinoseb was fed for
11 weeks to adult male Sherman rats (99 to 115 days of age) at dosage levels
of 75, 150, 225, or 300 pp*n. On the basis of food consurption data, the
authors calculated that these dosage levels averaged 3.8, 9.1, 15.6, or
22.2 rrg/kg/day, respectively. Both a normal control group and a pair-weight
control group (matched in body weight and pair-fed with the high-dose group)
were included in the study. Thirty-six rats were fed at dietary levels of 0
and 22.2 rrg/kg/day. All other groups consisted of 20 rats each. Four rats
from each of the groups fed 0 or 22.2 mg/kg were sacrificed after 10, 20, 30,
or 50 days of treatment. Half of the reminder in each group were sacrificed
V-23
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during the 11th week (71 to 77 days). The remaining rats were utilized for
reproductive and recovery studies. In rats fed 22.2 mg/kg/day, differential
classification of spermatozoa from the cauda epididymis indicated that 90% of
the sperm were atypical after 20 days of treatment. By 30 days, "bizarre" and
amorphous forms were found, and epididymal sperm counts were decreased.
Changes in the testes included abnormal spermatozoa and spermatids, multi-
nucleated spermatogenic cells at 20 and 30 days, and severe damage to sperma-
togenic cells by 50 days. Dietary levels of 225 or 300 ppm produced marked
oligospermia and extensive loss of spermatogenic cells in rats fed dinoseb for
11 weeks. Evidence of necrotic spermatogenic cells was seen in some tubules,
and only Sertoli cells remained in many tubules. Reproductive failure
occurred at 225 and 300 ppm, although mating behavior and libido appeared
normal.
Little or no recovery was seen during a 16-week period after 300-ppm
exposure was discontinued. The decrease in sperm count produced by doses of
150 or 225 ppm (9.1 or 15.6 mg/kg/day, respectively) appeared to be at least
partly reversible with time. Sperm counts of rats at the various dietary
levels after 71 to 77 days of exposure and after a 16-week recovery period are
shown in Table V-7. No effects of any type were seen in the animals fed
75 ppm (3.8 mg/kg/day).
In contrast, Osterloh et al. (1983) evaluated testicular toxicity of
dinoseb (purity of 98%) in male mice over a range of seven dose levels, and no
effects were seen in any of-the testicular parameters measured. Measurements
of testicular effect included sperm morphology, sperm count, and testicular
weight. Male hybrid (C57BL/6 x C3H)F, mice (7 to 10 weeks of age, four mice
per dose level) were injected intraperitoneally with 2.0, 4.3, 9.3, 20, or
43 mg/kg/day or administered commercial grade dinoseb (98% pure) in corn oil,
by gavage, at 20 mg/kg/day for 5 consecutive days. On day 35, the animals
were killed, and the testes and cauda epididymides were removed for study.
Total sperm counts were carried out on the epididymal sperm suspensions, and
portions were stained with Eosin Y for morphological examination. All mice
intraperitoneally administered 43 mg/kg/day died, and no effects on testicular
V-24
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Table V-7. Epidldymal Sperm Counts in Rats Fed Dinoseb for 71 to 77 Days
Weeks
after dis- Dietary
continuation level
of exposure (ppm)
0 0
75
150
225
300
0'
16 0
75
150
225
300
0'
Sperm content
of caudae and
Enididvmal fluid1
Rats
{n)
9
10
10
8
3
9
10
10
10
5
1
10
Epididymides
(n)
16
19
18
16
5
16
18
19
19
8
1
19
Sperm count6-'
(10/mg fluid)
1.45 +0.05
1.57 + 0.07
1.21 + 0.09d
0.05 + 0.02"
0"
1.54 ± 0.09
1.53 + 0.09
1.52 + 0.13
1.55 + 0.09
0.70 + 0.24"
0"
1.50 ± 0.06
vasa deferenti a
Rats
(n)
9
10
10
9
5
9
10
10
9
10
4
10
Sperm cells
(ioa)
379 + 40
458 + 28
206 + 1*
20 + 4d-'
9 -« 6
173 ± 29d
- 369 + 36
402 + 60
352 + 47
78 + 5"'f
6 + 6f
404 -i- 36
"Samples of less than 1 mg excluded; epididymal sample used as unit for
calculations.
"Values are group mean ± SEM.
BZero values do not indicate complete azoospermia, only that no sperm cells were
present-in enumerated squares.
"Differs from control (p <0.05).
"Pair-fed controls, given food equal to amount consumed by high-dose group.
'Adjusted count (adjusted for the weight of the caudae and vasa deferentia) differs
from control (p <0.05).
SOURCE: Adapted from Under et al. (1982).
V-25
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parameters were observed at the other dose levels. The authors suggested
several possibilities to account for the failure of ONBP to show effects in
this study. Although some dosage levels were comparable to those reported in
the study by Linder et al. (1982), the animals received only five daily doses,
as compared to continuous daily ingestion over an 11-week period in the Linder
report. In addition, since rats were used in the Linder study, species
differences may also have been a factor in production of varying results.
D. MUTAGENICITY
Relatively few studies investigating the genotoxic potential of dinoseb
have appeared in the published literature. This section includes -the pub-
lished experiments as well as a series of unpublished assays performed to meet
U.S. EPA registration requirements. These are categorized into genemutation
assays (Category 1), chromosome aberration assays (Category 2, none found for
dinoseb), and studies that assess other mutagenic mechanisms (Category 3).
The findings are discussed below.
1. Gene Mutation Assays (Category 1)
a. Reverse mutations in bacteria
Dinoseb (purity of 97.7%) elicited negative response when tested for
mutagenicity in the Ames assay employing four strains (TA1535, TA1537, TA1538,
and TA100) of Salmonella tvphimurium and one strain (WP2 uvrA") of Escherichia
coli (Simmon et a!., 1977). The assays were conducted in the absence or
presence of a metabolic activation system derived from an Aroclor 1254-
stimulated, rat liver homogenate. The Salmonella assays measured reversion to
histidine prototrophy, and the £. coli assays measured reversion of WP2 to
tryptophan independence.
Moriya et al. (1983) reported that dinoseb (purity not given) acetate was
not mutagenic in bacterial reversion assay systems with five strains (TA100,
TA98, TA1535, TA1537, and TA1538) of S. tvphimurium and one strain (WP2 her)
of E. coli. Dinoseb, along with 228 other pesticides, was tested with or
V-26
-------
without S9 mix at doses up to 5,000 ^g/plate (unless the compound showed
toxicity to bacteria at this dose).
Waters et al. (1982) tested dinoseb (purity not given) for mutagenic
potential in the Ames assay using $'. tvohimurium and f. coli and reported that
the herbicide was nonmutagenic. The assays were conducted both without and
with a metabolic activation system derived from Aroclor 1254-induced rat
livers. Five strains of i. tvphimurium (TA1535, TA1537, TA1538, TA98, and
TA100) and one strain of £. coli (WP2 uvrA) were used in these assays.
Dinoseb was tested along with a large number of other pesticides. Each
chemical was usually tested at a minimum of six concentrations; the highest
nontoxic concentration tested was 10 mg/plate unless the chemical -solubility
dictated otherwise.
b. Sex-linked recessive lethals fSLRL) in Drosoohila
Waters et al. (1982) reported that dinoseb (purity not given) did not
induce sex-linked recessive lethals in Drosoohila melanogaster. Doses tested
were not reported. A compound was considered nonmutagenic if it did not
elicit a 0.2% increase in mutation rate over the background and if the sample
population tested was sufficient to permit detection at the 95% confidence
level.
c. Mammalian cells in culture
Technical grade dinoseb, 4 to 48 jtg/mL without activation and 1.5 to
90 *g/mL with activation, was tested in the L5178Y mouse lymphoma cell line
for gene mutations (Den Boer, 1986). Without activation, small increases in
mutation frequencies were induced at higher concentrations, just exceeding the
minimum criteria for mutagenesis and thus indicating a weak mutagen. With
activation, large increases in mutation frequency accompanied by high toxicity
were noted. These increases could not be repeated but resulted in weakly
positive results. The authors, therefore, concluded the material to be weakly
positive.
V-27
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2. Other Genotoxic Effects (Category 31
a. Differential toxlcitv in bacteria
Dinoseb (purity of 97.7%) elicited positive response in DMA repair
synthesis assay using repair-deficient and repair-proficient strains of E.
£P_H (W3HO and p3478) and Bacillus subtiiU. (H17 and H45) (Simmon et al.,
1977). Both solvent and positive controls were run concurrently. Newell
(1981) reported that dinoseb (purity not given) was mutagenic in the DNA
repair synthesis assay with £. coli (polA) but not mutagenic with I. subtil is.
Waters (1982) reported that dinoseb (purity not given) was classified as
positive in three tests for primary DNA damage in prokaryotes. Differential
toxicity assays were conducted with £. coli strains p3478 (DNA polymerase I-
deficient polA") and W3100; B. subtilis strains M45 (recombination-deficient
recA") and H17; and S- tvphimurium strains SL4525 (rec*), SL4700 (rec'J,
TA1978, and TA1538. A positive reponse was indicated by a larger zone of
inhibition on a repair-deficient strain than on the normal strain. Doses
tested were not reported.
b. Mitotic recombination
Dinoseb (purity of 97.7%) was nonmutagenic in a mitotic recombination
assay with Saccharomvces cerevisiae D3 (Simmons et al., 1977). The compound
was tested at concentrations of 0.1, 0.2, and 0.3% (w/v-or v/v) either without
or with metabolic activation system from Aroclor 1254-induced rat livers.
Both positive and negative controls were run concurrently. Newell (1981) also
found dinoseb to be nonmutagenic in a mitotic recombination assay with
S. cerevisiae D3.
Waters et al. (1982) reported that dinoseb (purity not given) did not
induce mitotic recombination in S. cerevisiae 03. Five concentrations (not
specified) of the test chemical were tested both with and without metabolic
activation. A positive response was indicated by dose-related increases of
'V-28
-------
more than threefold in the absolute number of mitotic recombinants per .ml and
in the relative number of mitotic recombinations per 10* survivors.
c. Unscheduled DMA synthesis (UPS)
Dinoseb (purity of 97.7%) elicited negative response in the DOS assay
either without (dose range 1(T7 to 1Q~* M) or with (dose range 10" to 1(T3 M)
the metabolic activating system (Simmons et al., 1977). Five replicate
cultures of diploid WI-38 human fibroblast cells were used for the UDS assay.
Both solvent and positive controls were run concurrently. Mitchell (1981)
also reported that dinoseb (purity not given) was not mutagenic in the UDS
assay with diploid human fibroblasts (WI-38 cells); doses used were not
reported.
E. CARCINOGENICITY
In a 2-year feeding study conducted by Hazleton (1977), groups of
60 albino rats per sex were administered dinoseb at 0, 1, 3, or 10 mg/kg/day
in the diet. No increased incidences of tumors were found in high-dose rats
compared to controls. However, tissues from only 10 animals per sex from the
control and high-dose group in addition to the liver, kidneys, and lesions
from animals in the low- and mid-dose groups were examined at the interim
sacrifice (week 52) and terminal sacrifice (week 104).
Brown (1981) conducted a carcinogenicity study with groups of 70 male and
70 female CD mice administered 0, 1, 3, or 10 mg/kg/day of technical grade
dinoseb (purity not given) in the diet for 100 weeks.
Female mice showed an increased (but not dose-related) incidence of liver
adenomas and combined adenomas and carcinomas (Table V-8). Dinoseb induced
statistically significant (p <0.05) increases in liver adenomas in female mice
at the 3- and 10-mg/kg/day doses. The incidence for adenomas was
0/57, 3/59, 7/60, and 5/58 for the 0-, 1-, 3-, and 10-mg/kg/day doses, respec-
tively. Only one hepatocellular carcinoma was observed in female mice; this
occurred in the low-dose group. No adenomas or carcinomas were noted in the
V-29
-------
In rats administered dinoseb at dietary levels of 13.5 mg/kg/day for
21 days, blood urea nitrogen increased to 55 mg% versus 19.4 mg% in the
controls. This was accompanied with slight degenerative changes in .the renal
tubules and cloudy swelling in the liver. Dinoseb administered intraperi-
toneally at doses of 12 to 16 mg/kg/day for 5 days intensified inhibitory and
excitatory activities in the brains of rats, and daily doses of 2 to 8 mg/kg/-
day for 5 days were without effect.
Using the duckling as an experimental model, it has been demonstrated
that dinoseb, in common with a number of other dinitrophenols, has the ability
to produce cataracts following dietary exposure.
Dinoseb, administered at dietary levels of 300 ppm and above to rats for
60 days, resulted in a high incidence of death. Depressed growth was noted at
the lower dose levels (50 to 200 ppm) as were decreased organ weights and
lactic dehydrogenase and cholinesterase activities. Increases were seen in
organ-to-body weight ratios, alkaline phosphatase, alanine aminotransferase,
potassium, and urea nitrogen. Discrimination learning was not affected.
Diffuse tubular atrophy of the testes was. noted, especially at the 200-ppm
level.
In a 6-month feeding study, the body weights of rats receiving
5.4 mg/kg/day were slightly lower than those of controls at the end of the
treatment period. No changes were noted in hematology gross examination, mean
organ weights, and histopathology, except for a slight but statistically
significant increase in mean liver weight. Dietary levels of 13.5 mg/kg/day
resulted in increased mortality.
Beagle dogs administered 0.01 or 0.005% dinoseb in the diet for 91 days
were without adverse effects, and those administered 0.02 and 0.03% levels in
the diet showed decreased body weight gain, increased average liver weights,
mural endocarditis, and microscopic heart changes in females only. The NOAEL
was 0.01% (100 ppm), equivalent to 4 mg/kg/day.
V-32
-------
In a chronic toxicity study conducted in rats fed 1, 3, and 10 mg/kg/day
in the diet for 2 years, a compound-related decrease in mean thyroid weights
was reported. No other compound-related effects were observed; however,
histopathologic evaluation of tissues was conducted in only a limited number
of animals. The results suggest a LOAEL of 1 mg/kg/day.
Mice orally administered dinoseb in the. diet for 100 weeks, at 1, 3, and
10 mg/kg/day, showed cystic endometrial hyperplasia and atrophy, hyposperma-
togenesis, and testicular degeneration. Lenticular opacities were noted at
the 3- and 10-mg/kg/day dose levels; the lowest level was not examined for
this effect. A systemic NOAEL is less than 1 mg/kg/day.
Dinoseb has been found to cause skeletal anomalies in fetuses of several
species following oral, intraperitoneal, subcutaneous, and dermal administra-
tion to pregnant animals. Oral administration of dinoseb to mice on days 10
to 12 of gestation produced skeletal anomalies at 20 and 32 mg/kg/day;
maternal mortality was also present at these dose levels. However, in mice
orally administered 15 and 100 mg/kg during days 8 through 12 of gestation, no
effects were seen on postnatal parameters at day 22, 30, or 57. Other studies
suggest that the rat may be more susceptible than the mouse to the effects of
dinoseb. Pregnant Sprague-Dawley rats fed 8.6 mg/kg/day or more of dinoseb in
the diet on days 6 to 15 of gestation exhibited poor weight gain, sometimes
with ataxia, and lethargy. Fetal survival was decreased at and above doses of
6.9 mg/kg/day; decreases reached significance at or above 8.6 mg/kg/day.
Oral administration of dinoseb to mice on day 8 of gestation at doses of
26 and 33 mg/kg produced supernumerary ribs. The same finding was seen in
rats administered 10 mg/kg dinoseb on days 6 through 15 of gestation.
Skeletal anomalies were also observed in rabbits orally administered 10 mg/kg
dinoseb on days 6 through 18 of gestation, as were external and visceral
malformations.
Dinoseb appears to elicit an even greater incidence of developmental
anomalies after dermal exposure in pregnant rabbits. Increased incidences of
gross external, soft tissue, and skeletal malformations were noted in fetuses
V-33
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of rabbits percutaneously treated with dinoseb at 3 mg/kg/day or higher. These
malformations Included hydrocephaly, ralcrophthalmia, anophthalroia, cranio-
synostosis, and small eye sockets. The NOAEL for maternal toxlcity was also
1 mg/kg/day, based on mortality, slight decreases in body weight during the
dosing period, and increased incidences of gross lesions upon necropsy of
rabbits receiving dosages of 3 mg/kg/day or higher.
Treatment of pregnant mice with 17.7 mg/kg dinoseb administered intra-
peritoneally on days 10 to 12 of gestation resulted in a variety of fetal
defects, including fused or missing ribs, fused or missing sternebrae, fused
or unossified or absent vertebrae, and absent or unossified long bones.
Although doses of 10 to 15.8 mg/kg had no maternal or developmental effects
when administered on gestation days 10 to 12, 12.5 mg/kg/day on days 14 to 16
significantly increased resorption rates and reduced fetal weights.
In a study of postnatal morphology and functional capacity of kidneys In
neonates of Sprague-Dawley rats treated intraperitoneally with dinoseb, it was
demonstrated that approximately 40% of the fetuses of mothers intraperi-
toneally administered dinoseb at 8.0 to 9.0 mg/kg/day had dilated renal pelves
and/or ureters. Histological examination revealed relatively complete
recovery when offspring were examined at 6 weeks of age. In contrast, livers
of fetuses from this same group showed highly vacuolated cells on initial
examination; these toxic effects were still present in the livers of the
offspring 6 weeks later, along with necrotic cells and pyknotic or karyorrhec-
tic nuclei in other cells. Thus, the liver showed little evidence of recovery
from the initial damage.
It has been found that pretreatment of pregnant mice with SKF-525A (a
mixed-function oxidase inhibitor) potentiates resorptions and reductions in
fetal body weights induced by dinoseb when injected intraperitoneally. In
contrast, pretreatment with phenobarbital, which stimulates the hepatic mixed
function oxidase system, inhibits these effects.
In rat studies in which dinoseb was administered in the diet for five
consecutive generations at 1, 3, and 10 mg/kg/day, no effects on survival,
V-34
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fecundity, or fertility were seen. At 10 mg/kg/day, the litter sizes at birth
and the pup weights at weaning were reduced; this was attributed to maternal
toxicity.
Dietary levels of dinoseb at 15.6 or 22.2 mg/kg/day for 11 weeks produced
marked oligospermia and extensive loss of spermatogenic cells in the testes of
rats. Little recovery occurred during the 16 weeks following cessation of
exposure. At a dose level of 9.1 mg/kg/day, decreased epididymal sperm
counts, atypical epididymal spermatozoa, and minimal testicular changes were
present. These effects appeared to be reversible with time. No effects were
seen in the rats fed 3.8 mg/kg/day in this 11-week study.
No testicular effects were noted, however, in mice receiving oral or
intraperitoneal doses of up to 20 mg/kg/day for 5 consecutive days. Intra-
peritoneal doses of 43 mg/kg/day were lethal.
One report suggested that orally administered dinoseb (about 20 mg/kg)
may have long-term inhibitory effects on both the cellular and humoral immune
responses in the hamster.
A number of assays were conducted to determine the mutagenic potential of
dinoseb. Negative responses were elicited in the Ames assay with
S. tvphimurium and E. coli. sex-linked recessive lethal assay in fi. melano-
gaster. mitotic recombination assay in £. cerevisiae. and unscheduled DNA
synthesis assay in human fibroblasts. However, positive responses were
elicited in DNA repair synthesis assays using repair-deficient and repair-
proficient strains of I. coli. B. subtilis. and S. tvohimurium. Dinoseb also
induced small increases in mutation frequencies in a mouse lymphoma cell line.
No increases in tumor incidences were observed in rats fed dinoseb at
levels of 0, 1, 3, or 10 mg/kg/day in the diet for 104 weeks. However, only a
limited number of animals were examined histologically. Mice administered
dinoseb orally in the diet for 100 weeks at 1, 3, and 10 mg/kg/day showed
equivocal oncogenic effects, although statistically significant increases in
V-35
-------
the incidence of liver adenomas and combined adenomas and carcinomas were
observed in female mice only.
V-36
-------
VI, HEALTH EFFECTS IN HUMANS
A. CLINICAL CASE STUDIES
Smith (1981) reported a case history of an individual apparently poisoned
by a dinoseb-containing herbicide. A self-employed farmer unfamiliar with use
of the herbicide sprayed an area of new grass seed with the product. During
spray operations, he unplugged a spray jet with his bare hands. He wore a
gauze face mask, which he noted was heavily stained yellow at the end of the
spraying operation. Later that day, the farmer developed a headache, malaise,
lassitude, and sweating. The next day, he sought medical advice at the
nearest hospital casualty department. After a conference with a member of the
manufacturer's medical department, and in view of the minimal exposure and the
clinical profile, the joint tentative diagnosis was that the patient was
suffering from influenza, and he was referred to the care of his general
practitioner.
Over the subsequent 5 days, the patient had anorexia, bouts of excessive
sweating and intermittent shivering, pains in the chest and abdomen, excessive
thirst, restlessness, insomnia, loose stool, and weight loss (10 kg during the
week). He developed further symptoms of respiratory involvement, including
shortness of breath and hemoptysis, and displayed personality changes that
alarmed the family. Six days after the incident, the farmer was seen by his
general practitioner, who referred him to the hospital where he was admitted.
On admission, the patient was flushed, with a temperature of 39.8°C. He
exhibited intermittent dyspnea, spasmodic coughing, dullness at the base of
one lung, and crepitations. He was immediately treated with oxytetracycline.
The patient complained of photophobia and some neck stiffness and was found to
have a positive Kernig sign. His erythrocyte sedimentation rate was
96 mm/hour. The urine was discolored yellow. A blood sample was collected,
analyzed for dinitro compounds, and found to be negative. Liver function was
impaired, and chest X-ray revealed patchy shadowing at the bases. Lung
function tests indicated considerable impairment.
VI-1
-------
At the end of 1 week,-the farmer's clinical picture had improved suffi-
ciently to permit his discharge from the hospital; however, his liver function
test was more abnormal than it had been at the time of admission. Two weeks
later, as an outpatient, he was still complaining of lethargy, night sweats,
and forgetfulness. His condition slowly improved, and after 10 to 12 weeks he
was symptom free. However, 6 months after the incident, his blood urea was
reported to be 7.9 mmol/L (the normal range is 3.5 to 6.5 mmol/L). The author
of this report suggested that both inhalation and skin exposure may have
played a role in the toxicity. It is interesting to note that in animal
studies by McCormack et a!. (1980) (see Section V.C.l.b), offspring of
dinoseb-exposed rats showed kidney damage from which they recovered by 42 days
postpartum, but liver damage had not improved and may have been worse. This
finding is consistent with the case study reported above.
Heyndrickx et al. (1964) reported a fatal human exposure to two herbi-
cides, Nitrader 40 (40% dinitro-ortho-cresol) and Dinorsol PL (14% dinoseb).
Five days after spraying with these two herbicides, a farm worker suddenly
became ill. He vomited frequently and felt tired. The following day he
complained of violent spasms, intense thirst, and tachycardia. He was
hospitalized, and he died the following day. At autopsy, no specific cause of
death could be ascertained. Dinitro-ortho-cresol was identified from the skin
of the hand but could not be identified in other tissues. An unidentified
metabolite was found in urine. Both herbicides could be identified in
extracts of the overalls, cap, and mask. The authors point out that delayed
effects in humans have been previously reported for dinitro-ortho-cresol
poisoning. The contribution of dinoseb to the toxic response in this incident
cannot be determined, although the analysis of clothing indicated that about
twice as much dinoseb as dinitro-ortho-cresol was present at the time of
analysis.
B. EPIDEMIOLOGICAL STUDIES
No epidemic!ogical studies of dinoseb were found.
VI-2
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more than threefold in the absolute number of mitotic recombinants per ml and
in the relative number of mitotic recombinations per 10s survivors.
c. Unscheduled DNA synthesis (UPSI
Dinoseb (purity of 97.7%) elicited negative response in the UDS assay
either without (dose range 10"7 to KT4 M) or with (dose range 10"5 to 10'3 M)
the metabolic activating system (Simmons et al., 1977). Five replicate
cultures of diploid MI-38 human fibroblast cells were used for the UOS assay.
Both solvent and positive controls were run concurrently. Mitchell (1981)
also reported that dinoseb (purity not given) was not mutagenic in the UDS
assay with diploid human fibroblasts (WI-38 cells); doses used were not
reported.
E. CARCINOGENICITY
In a 2-year feeding study conducted by Hazleton (1977), groups of
60 albino rats per sex were administered dinoseb at 0, 1, 3, or 10 mg/kg/day
in the diet. No increased incidences of tumors were found in high-dose rats
compared to controls. However, tissues from only 10 animals per sex from the
control and high-dose group in addition to the liver, kidneys, and lesions
from animals in the low- and mid-dose groups were examined at the interim
sacrifice (week 52) and terminal sacrifice (week 104).
Brown (1981) conducted a careinogenicity study with groups of 70 male and
70 female CD mice administered 0, 1, 3, or 10 mg/kg/day of technical grade
dinoseb (purity not given) in the diet for 100 weeks.
Female mice showed an increased (but not dose-related) incidence of liver
adenomas and combined adenomas and carcinomas (Table V-8). Dinoseb induced
statistically significant (p <0.05) increases in liver adenomas in female mice
at the 3- and 10-mg/kg/day doses. The incidence for adenomas was
0/57, 3/59, 7/60, and 5/58 for the 0-, 1-, 3-, and 10-mg/kg/day doses, respec-
tively. Only one hepatocellular carcinoma was observed in female mice; this
occurred in the low-dose group. No adenomas or carcinomas were noted in the
V-29
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Table V-8. Incidence of Hepatocellular Adenoma and Carcinoma in Nice
Receiving Dirioseb in the Diet for 100 Weeks
Dosaae level fma/ka)
0 if contrail 1
Neopl asm
No. of livers
examined
Hepatocellular
adenoma
Hepatocellular
carcinoma
M
70
11
5
F M
70 70
0 16
0 4
3
F M
70 70
3 17
1 9
10
F M
70 70
7 16
0 5
'
F
70
5
0
SOURCE: Adapted from Brown (1981).
V-30
-------
female controls. No statistical difference from controls was observed in the
incidence of hepatocellular adenoma and carcinoma in males at any level. No
biological significance was attributed to the increased occurrence of these
adenomas in females, since there was no dose relationship, only a low number
of animals was affected, and there was a lack of other hepatocellular changes
commonly associated with carcinogens. In addition, the lack of any adenomas
in the female controls is not consistent with the normal incidence in controls
of this strain. It was concluded that there were no treatment-related
f
neoplastic changes.
Further details on both studies were described in Section V.B.2, Chronic
Effects.
In a separate screening study, mice failed to demonstrate any significant
increase in tumors (Innes et al., 1969). Two strains of mice {hybrids of
female C57BL/6 and male C3H/Anf or AKR mice, 18/sex/group) were exposed to
dinoseb for 18 months. The animals were first exposed via gavage at
2.15 mg/kg/day for 3 weeks beginning at 1 week of age; then they were fed a
diet containing 7 ppm dinoseb (1.05 mg/kg/day) throughout the observation
period of approximately 18 months. Equal numbers of mice served as controls.
After 18 months on diet, dinoseb did not cause any significant increase in
tumors in mice.
F. SUMMARY
Acute oral LDSO values for the rat, mouse, rabbit, and guinea pig range
from 14 to 114 mg/kg. The intraperitoneal LDSO value has been reported as
20.2 mg/kg for female mice and 10 mg/kg for male mice. Prostration, rapid
respiration, and convulsions immediately preceding death were observed in
guinea pigs receiving acute doses. Elevated environmental temperatures
lowered the LDSO for female mice intraperitoneally injected with dinoseb.
Dinoseb is apparently well absorbed through the intact skin, with dermal LDSO
values in the rat ranging from 67 to 134 mg/kg; absorption depended to a large
extent on the method of application and the covering employed.
V-31
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In rats administered d.inoseb at dietary levels of 13.5 mg/kg/day for
21 days, blood urea nitrogen increased to 55 mg% versus 19.4 mg% in the
controls. This was accompanied with slight degenerative changes in .the renal
tubules and cloudy swelling in the liver. Dinoseb administered intraperi-
toneally at doses of 12 to 16 mg/kg/day for 5 days intensified inhibitory and
excitatory activities in the brains of rats, and daily doses of 2 to 8 mg/kg/-
day for 5 days were without effect. .
Using the duckling as an experimental model, it has been demonstrated
that dinoseb, in common with a number of other dinitrophenols, has the ability
to produce cataracts following dietary exposure.
Dinoseb, administered at dietary levels of 300 ppm and above to rats for
60 days, resulted in a high incidence of death. Depressed growth was noted at
the lower dose levels (50 to 200 ppm) as were decreased organ weights and
lactic dehydrogenase and cholinesterase activities. Increases were seen in
organ-to-body weight ratios, alkaline phosphatase, alanine aminotransferase,
potassium, and urea nitrogen. Discrimination learning was not affected.
Diffuse tubular atrophy of the testes was noted, especially at the 200-ppm
level.
In a 6-month feeding study, the body weights of rats receiving
5.4 mg/kg/day were slightly lower than those of controls at the end of the
treatment period. No changes were noted in hematology gross examination, mean
organ weights, and histopathology, except for a slight but statistically
significant increase in mean liver weight. Dietary levels of 13.5 mg/kg/day
resulted in increased mortality.
Beagle dogs administered 0.01 or 0.005% dinoseb in the diet for 91 days
were without adverse effects, and those administered 0.02 and 0.03% levels in
the diet showed decreased body weight gain, increased average liver weights,
mural endocarditis, and microscopic heart changes in females only. The NOAEL
was 0.01% (100 ppm), equivalent to 4 mg/kg/day.
V-32
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C. HIGH-RISK POPULATIONS
No specific subpopulations were identified that appear to be at greater
risk from dinoseb exposure than the general population. Certain work popu-
lations, including those involved in manufacture and, perhaps to a greater
extent, those who mix and apply this herbicide in the field, have the poten-
tial for greater exposure.
D. SUMMARY
One clinical case history of dinoseb poisoning, from which the patient
apparently recovered, has been reported in the literature. Elevated body
temperature, liver damage, and subsequent lung involvement were the major
effects. The liver damage appeared to be particularly long lasting. This has
also been reported in experimental animals (see Chapter V of this report).
No relevant epidemiological studies were found, and no high-risk
populations were identified.
VI-3
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-------
VII. MECHANISMS OF TOXICITY
A. UNCOUPLING OF OXIDATIVE PHOSPHORYLATION
Ilivicky and Casida (1969) reported an extensive investigation of the
uncoupling of oxidative phosphorylation in mitochondria by five dinitrophenol
compounds, including dinoseb. Mitochondria were prepared from the brains and
livers of male albino mice (18 to 20 g, strain unspecified). The potency of
the uncoupling action was judged by the minimum concentration of chemical
necessary for complete uncoupling. Brain mitochondria were up to 50-fold more
sensitive to the uncoupling action of the 2,4-dinitrophenols than were liver
mitochondria. The minimum uncoupling concentration for dinoseb with mouse
liver mitochondria was 1.0 MM compared with 50 *M for 2,4-dinitrophenol. With
brain mitochondria, the differences were 0.5
-------
B. METHEMOGLOBIN FORMATION
Dlnoseb produces considerable methemoglobin in ruminants, although
methemoglobin formation is minimal in other species exposed to the herbicide
{Froslie and Karlog, 1970; Froslie, 1974, 1976J. These authors suggested that
methemoglobin formation is related to the formation of the diamino metabolite
of dinoseb in the rumen of these animals. Froslie (1976) found that elevated
methemoglobin levels produced in seven sheep after intraruminal administration
of nitrite lasted only about 12 hours, and dinoseb-induced methemoglobinemia
persisted for at least 2 to 3 days. The dinoseb effect correlated with a
progressive and almost complete inhibition of NADH-methemoglobin reductase in
the red blood cells. This inhibition may underlie the methemoglobinemia found
in the ruminant after ingestion of dinoseb.
C. INTERACTIONS
In a study discussed in detail in this report (Section V.C.l.b), Preache
and Gibson (1975b) found that SKF-525A potentiated the resorptions and
reductions in fetal body weight induced by dinoseb. In contrast, pretreatment
of pregnant rats with phenobarbital inhibited these effects.
D. SUMMARY
Dinoseb, like other dinitrophenols, is an uncoupler of oxidative
phosphorylation, particularly in brain mitochondria. Dinoseb inhibition of
brain oxidative phosphorylation correlates with signs and severity of toxi-
city. In animals showing severe external signs of poisoning, brain mito-
chondria! oxidative phosphorylation was completely inhibited.
Synergistic effects have been shown with SKF-525A, and antagonistic
effects have been shown with phenobarbital following intraperitoneal adminis-
tration in pregnant Swiss-Webster mice. The former potentiates and the latter
inhibits resorptions and reductions in fetal body weights induced by dinoseb.
VII-2
-------
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
The quantification of toxicological effects of a chemical consists of an
assessment of noncarcinogenic and carcinogenic effects. Chemicals that do not
produce carcinogenic effects are believed to have a threshold dose below which
no adverse, noncarcinogenic health effects occur, whereas carcinogens are
assumed to act without a threshold. The quantification of noncarcinogenic
effects (One-, Ten-, Longer-term Health Advisories) that were calculated using
suitable oral toxicity studies are presented in this chapter.
A. PROCEDURES FOR QUANTIFICATION OF TOXICOLOGICAL EFFECTS
1. Noncarcinoqenic Effects
In the quantification of noncarcinogenic effects, a Reference Dose (RfD),
formerly called the Acceptable Daily Intake (ADI), is calculated. The RfD is
an estimate of a daily exposure to the human population that is likely to be
without appreciable risk of deleterious health effects, even if exposure
occurs over a lifetime. The RfD is derived from a No-Observed-Adverse-Effect
Level (NOAEL) or Lowest-Observed-Adverse-Effect Level (LOAEL), identified from
a subchronic or chronic study, and divided by an uncertainty factor (UF) . The
RfD is calculated as follows:
Rfn = . fNOAEL or IQAELl = mg/kg bw/day
*TU Uncertainty factor -- *' y
Selection of the uncertainty factor to be employed in the calculation of
the RfD is based on professional judgment while considering the entire. data
base of toxicological effects for the chemical. To ensure that uncertainty
factors are selected and applied in a consistent manner, the Office of
Drinking Water employs a modification to the guidelines proposed by the
National Academy of Sciences (NAS, 1977, 1980), as follows:
VIII-1
-------
An uncertainty factor of 10 is generally used when good chronic or
subchronic human exposure data identifying a NOAEL are available and
are supported by good chronic or subchronic toxicity data for other
species.
An uncertainty factor of 100 is generally used when good chronic
toxicity data identifying a NOAEL are available for one or more animal
species (and human data are not available), or when good chronic or
subchronic toxicity data identifying a LOAEL in humans are available.
An uncertainty factor of 1,000 is generally used when limited or
incomplete chronic or subchronic toxicity data are available, or when
good chronic or subchronic toxicity data identifying a LOAEL, but not
a NOAEL, for one or more animal species are available.
The uncertainty factor used for a specific risk assessment is based prin-
cipally on scientific judgment, rather than scientific fact, and accounts for
possible intra- and interspecies differences. Additional considerations not
incorporated in the NAS/ODW guidelines for selection of an uncertainty factor
include the use of a less-than-lifetime study for deriving an RfD, the
significance of the adverse health effect, and the counterbalancing of
beneficial effects.
From the RfD, a Drinking Water Equivalent Level (DWEL) can be calculated.
The DWEL represents a medium-specific {i.e., drinking water) lifetime exposure
at which adverse, noncarcinogenic health effects are not expected to occur.
The DWEL assumes 100% exposure from drinking water. The DWEL provides the
noncarcinogenic health effects basis for establishing a drinking water
standard. For ingestion data, the DWEL is derived as follows:
DWEL =» RfD x (body weight in kg)
Drinking water volume in L/day
mg/L
where:
Body weight * assumed to be 70 kg for an adult.
VIII-2
-------
Drinking water volume = assumed to be 2 L per day for an adult.
In addition to the RfD and the DWEL, Health Advisories (HAs) for
exposures of shorter duration (One-day, Ten-day, and Longer-term HAs) are
determined. The HA values are used as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
The HAs are calculated using a similar equation to the RfD and DWEL; however,
the NOAELs or LOAELs are identified from acute or subchronic studies. The HAs
are derived as follows:
HA - (NOAEL or LOAEL) x fbw) - mg/L { <.g/L)
(_ L/day) x (UF)
Using the above equation, the following drinking water HAs are developed
for noncarcinogenic effects:
1. One-day HA for a 10-kg child ingesting 1 L water per day.
2. Ten-day HA for a 10-kg child ingesting 1 L water per day. .
3. Longer-term HA for a 10-kg child ingesting 1 L water per day.
4. Longer-term HA for a 70-kg adult ingesting 2 L water per day.
The One-day HA, calculated for a 10-kg child, assumes a single acute
exposure to the chemical and is generally derived from a study of less than
7 days' duration. The Ten-day HA assumes a limited human exposure period of
1 to 2 weeks and is generally derived from a study of less than 30 days'
duration. The Longer-term HA is calculated for both a 10-kg child and a 70-kg
adult and assumes a human exposure period of approximately 7 years (or 10% of
an individual's lifetime). The Longer-term HA is generally derived from a
study of subchronic duration (exposure for 10% of an animal's lifetime).
VIII-3
-------
2. Carcinogenic Effects
The EPA categorizes the carcinogenic potential of a chemical, based on
the overall weight of evidence, according to the following scheme:
Group A: Known Human Carcinooen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of carcino-
genicity in animals with limited (Group Bl) or inadequate
(Group B2) evidence in humans.
Group C: Possible Human Carcinogen. Limited evidence of carcinogeni-
city in animals in the absence of human data.
. Group D: Not Classified as to Human Carcinogenicitv. Inadequate human
and animal evidence of carcinogenicity or for which no data
are available.
Group E: Evidence of Noncarcinogem'citv for Humans. No evidence of
carcinogenicity in at least two adequate animal tests in
different species or in both adequate epidemiologic and
animal studies.
If toxicological evidence leads to the classification of the contaminant
as a known, probable, or possible human carcinogen, mathematical models are
used to calculate the estimate of excess cancer risk associated with the
ingestion of the contaminant in drinking water. The data used in these
estimates usually come from lifetime exposure studies in animals. To predict
the risk for humans from animal data, animal doses must be converted to
equivalent human doses. This conversion includes correction for noncontinuous
exposure, less than-lifetime studies, and for differences in size. The factor
that compensates for the size difference is the cube root of the ratio of the
VIII-4
-------
animal and human body weights. It is assumed that the average adult human
body weight Is 70 kg and that the average water consumption of an adult human
Is 2 liters of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure via
ingestion of water. The cancer unit risk is usually derived from a linearized
multistage model, with a 95% upper confidence limit providing a low-dose
estimate; that is, the true risk to humans, while not identifiable, is not
likely to exceed the upper limit estimate and, in fact, may be lower. Excess
cancer risk estimates may also be calculated using other models such as the '
one-hit, Weibull, logit, and probit. There is little basis in the current
understanding of the biological mechanisms involved in cancer to suggest that
any one of these models is able to predict risk more accurately than any
others. Because each model is based on differing assumptions, the estimates
that were derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an inherent uncertainty due to the systematic and
random errors in scientific measurement. In most cases, only studies using
experimental animals have been performed. Thus, there is uncertainty when the
data are extrapolated to humans. When developing cancer risk rate levels,
several other areas of uncertainty exist, such as the incomplete knowledge
concerning the health effects of contaminants in drinking water; the impact of
the experimental animal's age, sex, and species; the nature of the target
organ system(s) examined; and the actual rate of exposure of the internal
targets in experimental animals or humans. Dose-response data usually are
available only for high levels of exposure, not for the lower levels of
exposure closer to where a standard may be set. When there is exposure to
more than one contaminant, additional uncertainty results from a lack of
information about possible synergistic or antagonistic effects.
VIII-5
-------
B. QUANTIFICATION OF NONCARCINOGENIC EFFECTS FOR OINOSEB
The following Health Advisories (One-day, Ten-day, and Longer-term), the
Reference Dose, and the Drinking Water Equivalent Level were calculated based
on ingestion data from suitable short- and long-term oral toxicity studies.
1. One-dav Health Advisory
No suitable studies were found for calculating the One-day Health
Advisory. U.S. EPA (1987J recommended that the Ten-day HA value of 0.3 tag/I
for a 10-kg child (calculated below) be used as a conservative estimate of the
One-day HA value.
2. Ten-day Health Advisory
In a developmental toxicity study with rabbits (Becker, 1986b), the oral
administration of dinoseb (purity 98.1%) produced neural tube defects at doses
greater than 3 mg/kg/day (NOAEL). The results of a recently completed
developmental toxicity study {Hoberman, 1987), in which pregnant rabbits
received percutaneous dosages of dinoseb, identified a developmental NOAEL of
1 mg/kg/day, based on increased incidences of gross external, soft tissue, and
skeletal malformations in fetuses from dams receiving dosages of 3 mg/kg/day
or higher. Although the Hoberman study (1987) identified a lower NOAEL, the
oral study represents the more appropriate route of exposure. Data from an
absorption study in monkeys (Bucks, 1987) suggest that a percutaneous dose of
0.2 mg/cm2 results in the maximum absorption rate and higher doses do not
result in greater absorption. Furthermore, a dermal absorption model
developed by the Office of Drinking Water indicates that human exposure to
dinoseb in the drinking water via the dermal route is negligible. -Therefore,
the developmental toxicity study of Becker (1986b) was selected as the basis
for determination of the Ten-day HA. While 1t is reasonable to base the Ten-
day HA for an adult on a positive developmental toxicity study, there is some
question as to whether it is appropriate to base the Ten-day HA for a 10-kg
child on such a study. However, since this study is of appropriate duration
VIII-6
-------
and since the fetus may be more sensitive than a 10-kg chiId, it was judged
that, while this nurber may be overly conservative, it is a reasonable basis
for the Ten-day HA for a 10-kg chi Id.
Using a t&EL of 3 rrg/kg/day, the Ten-day HA for a 10-kg chi Id is cal-
culated as follows:
Ten-day HA = (3 rro/kQ/dav) (10 kg) = 0.3 nrg/L (300 f/g/L)
(100) (1 L/day)
where:
3 rrg/kg/day = NZPEL, based on the absence of teratogenic effects
in rabbits.
10 kg = assured body weight of a child.
100 = uncertainty factor, chosen in accordance with
IsttS/OWguidel ines for use with a rOEL from an
animal study.
1 L/day = assured daily water consumption of a child.
3. Longer - te rm Hea I th Adv i sory
The Hall et al. (1978) 153-day dietary dinoseb (purity 80%) study in rats
was originally selected to serve as the basis for determination of the Longer-
term l-A (decreased growth was observed at all exposure levels with a LD4EL of
2.5 mg/kg/day). Subsequently, however, a 3-generation reproduction study in
rats (Irvine and Amnitage, 1981) identified a l&EL of 1 mg/kg/day (purity
98.4%), based on decreases in pup body weights at all dose levels. Since a
reproduction study is of appropriate duration, the Irvine and Armitage (1981)
study has been selected to serve as the basis for determination of the Longer-
term m.
Using a LQaEL of 1 rrg/kg/day, the Longer-term m for a 10-kg chid is
calculated as follows:
VI11-7
-------
Longer-term HA
fl mq/ko/davUlO ko) - 0.010 mg/L (10
(1,000)(1 L/day)
where:
1 mg/kg/day
10 kg
1,000
LOAEL, based on decreased pup body weight.
assumed body weight of a child.
uncertainty factor, chosen in accordance with
NAS/ODW guidelines for use with a LOAEL from an
animal study.
1 L/day = assumed daily water consumption of a child.
The Longer-term HA for a 70-kg adult is calculated as follows:
Longer-term HA
fl mq/ko/davH70 kal
(1,000)(2 L/day)
0.035 mg/L (40 »g/L)
where:
1.0 mg/kg/day
70 kg
1,000
LOAEL, based on decreased pup body weight.
assumed body weight of an adult.
uncertainty factor, chosen in accordance with
NAS/ODW guidelines for use with a LOAEL from an
animal study.
2 L/day assumed daily water consumption of an adult.
4- Reference Dose and Drinking Water Equivalent Level
Originally, the 2-year dietary study in rats by Hazleton (1977) was
selected to serve as the basis for determination of the Lifetime HA. In this
study, a LOAEL of 1 mg/kg/day was identified based on a compound-related
decrease in mean thyroid weights in all dosed males. However, tissues from
only a limited number of animals were examined njstopathologically. A more
VIII-8
-------
conrplete histopatho logical exani nation of tissues from mice fed diets contain-
ing dinoseb for 100 weeks (Brown, 1981) also identified a ID4EL of 1 mg/kg/-
day. Cystic endometriaf hyperplasia and atrophy, hypospenrrBtogenesis, and
degeneration of the testes were noted in females and males, respectively,
receiving 1, 3, or 10 nrg/kg/day. This LQAEL of 1 rrg/kg/day was also supported
by a three-generation reproductive study (Irvine and Armitage, 1981) which
demonstrated decreased fetal weights and a decrease in pup body weights at all
dose levels. Using a LQ4EL of 1 rrg/kg/day, the RfD for a 70-kg adult is
calculated as follo/vs:
Step 1: Determination of the Reference Dose (RfD)
RfD = (1 rra/kQ/dav) = 0.001 rrg/kg/day
(1,000)
where:
1 rrg/kg/day = LO4EL, based on decreased thyroid weight in male rats and/or
hypospermatogenesis, and degeneration of the testes and
cystic endorretrial hyperplasia and atrophy in male and female
mice in chronic dietary studies and decreased fetal weights
and a decrease in pup body weights in a three-generation
reproductive study.
1,000 = uncertainty factor, chosen in accordance with NQS/OWguide
I ines for use with a UCWEL from an animal study.
Step 2: Determination of the Drink ing V\ater Equivalent Level (DttEL)
OAEL = (0.001 nrq/kQ/dav) (70 kol - 0.035 rrg/L (40 //g/L)
(2 L/day)
where:
0.001 rrg/kg/day = RfD.
70 kg = assured body weight of an adult.
2 L/day = assured daily water consumption of an adult.
VI11-9
-------
-------
IX. REFERENCES
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IX-1
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Ernst W. 1968. Metabolism of substituted dinitrophenols and ureas in mammals
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IX-2
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Industrial Bio-Test Laboratories, Inc. 1966. Summary of acute studies with
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Newell G. 1975. In vitro and in vivo studies of selected pesticides to
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